Proteins for the treatment of epithelial barrier function disorders

ABSTRACT

The disclosure related to therapeutic proteins and pharmaceutical compositions comprising said proteins, which have utility in treating various human diseases. In particular aspects, the disclosed therapeutic proteins are useful for treating human gastrointestinal inflammatory diseases and gastrointestinal conditions associated with decreased epithelial cell barrier function or integrity. Further, the disclosed therapeutic proteins are useful for treating human inflammatory bowel disease, including inter alia, Crohn&#39;s disease and ulcerative colitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityof U.S. patent application Ser. No. 16/614,226, filed on Nov. 15, 2019,which is a National Stage Application under 35 U.S.C. § 371 and claimsthe benefit of International Application No. PCT/US2018/033347, filed onMay 18, 2018, which claims the benefit of priority to U.S. ProvisionalApplication No. 62/508,501 filed on May 19, 2017, the contents of whichare hereby incorporated by reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 47192-0013002_SL_ST26.xml. The XMLfile, created on Aug. 27, 2023, is 19,059 bytes in size. The material inthe XML file is hereby incorporated by reference in its entirety.

FIELD

The present disclosure relates to novel proteins and pharmaceuticalcompositions comprising said proteins that have application, inter alia,in the treatment of gastrointestinal inflammatory diseases andepithelial barrier function disorders. In some embodiments, the proteinsand pharmaceutical compositions described herein have particularapplication in the treatment or prevention of disease states associatedwith abnormally permeable epithelial barriers as well as inflammatorybowel diseases or disorders.

BACKGROUND

Inflammatory bowel disease (IBD) is a heterogeneous disease of unknownetiology resulting in frequent and bloody bowel movements accompaniedwith histopathological damage to the gastrointestinal mucosa (Zhang etal., 2017, Front Immunol, 8:942). While specific triggers of diseaseremain poorly defined, one proposal of disease progression suggests abreakdown of intestinal barrier function allows bacteria or bacterialcomponents to translocate into mucosal tissue (Coskun, 2014, Front Med(Lausanne), 1:24; Martini et al., 2017, Cell Mol Gastroenterol Hepatol,4:33-46). Bacterial translocation results in activation of inflammatorysignaling which triggers additional barrier disruption, resulting in acyclic amplification loop of barrier disruption, bacterial translocationand inflammation. While many current therapies target inflammation, thelack of therapies promoting mucosal healing provides an opportunity fornovel therapies promoting epithelial repair and intestinal barrierintegrity.

Expanding upon the hypothesis that bacterial translocation can triggerIBD, more recent studies have demonstrated detrimental changes inintestinal microbiota, or dysbiosis, may promote development of IBD.

Currently, many IBD therapeutics available in the market merely aim totarget and suppress the discussed inflammatory response associated withIBD. While helpful, this narrow therapeutic mode of action disregardsthe important contribution that epithelial barrier integrity plays inthe etiology of the disease.

Thus, there is a great need in the art for the development of atherapeutic, which not only suppresses the immune system's inflammatoryresponse, but that also acts in concert to restore the epithelialbarrier function in an individual. Also, there is a need for theproduction of a protein therapeutic such as that described herein whichis stable through the manufacturing and/or processing of the proteintherapeutic as well as under long term storage conditions.

SUMMARY OF THE DISCLOSURE

The present disclosure addresses the important need in the medicalcommunity for a therapeutic which can effectively treat a subjectsuffering from a gastrointestinal disorder such as an inflammatory boweldisease (IBD). In one aspect, novel protein therapeutics are providedwhich can maintain epithelial barrier integrity and/or improveepithelial barrier repair. In some embodiments, the epithelial barrieris intestinal epithelial barrier. These protein therapeutics can alsoreduce inflammation of the intestine of the subject and/or decreasesymptoms associated with inflammation of the intestine.

The protein therapeutics provided herein are useful in treating thenumerous diseases and/or symptoms that may be associated with decreasedgastrointestinal epithelial cell barrier function or integrity.

In some embodiments, the disclosure teaches novel protein therapeuticsderived from the microbiome and methods of utilizing said proteintherapeutics. In a particular embodiment, the novel protein therapeuticcomprising a therapeutic protein (SG-14) derived from the microbiome,wherein the therapeutic protein comprises an amino acid sequence havingat least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, or 100%, sequence identity to the polypeptidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 and SEQ ID NO:7, is provided. In some embodiments, theprotein does not comprise an amino acid sequence identical to SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. In yet other embodiments,the protein comprises an amino acid sequence which is not naturallyoccurring.

In some embodiments, the therapeutic protein comprises the amino acidsequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 or afragment or variant thereof. In other embodiments, the protein comprisesthe amino acid sequence of SEQ ID NO:3. In still other embodiments, theprotein comprises the amino acid sequence of SEQ ID NO:5. In yet otherembodiments, the protein comprises the amino acid sequence of SEQ IDNO:7.

In some embodiments, the therapeutic protein comprises an amino acidsequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to SEQ ID NO:3, wherein the amino acid sequence has at least1, 2, 3 or 4 amino acid substitutions relative to SEQ ID NO:1 or to SEQID NO:3. In other embodiments, the amino acid sequence has at least 2and less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35amino acid substitutions relative to SEQ ID NO:3. In still otherembodiments, the therapeutic protein comprises an amino acid sequencewhich is not naturally occurring.

In some embodiments, the therapeutic protein is about 500 to 700 aminoacids, 550 to 650 amino acids, 600 to 650 amino acids, 615 to 650 aminoacids, 625 to 640 amino acids, 625 to 635 amino acids, 650 to 700 aminoacids, or 660 to 670 amino acids in length. In other embodiments, thetherapeutic protein is 625, 626, 627, 628, 629, 630, 631, 632, 633, 634,634, 636, 637, 638, 660, 661, 662, 663, 664, or 665 amino acids inlength.

In some embodiments, the therapeutic protein comprises an amino acidsequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to SEQ ID NO:7, wherein the amino acid sequence has at least1, 2, 3 or 4 amino acid substitutions relative to SEQ ID NO:3 or to SEQID NO:7. In other embodiments, the amino acid sequence has at least 2and less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35amino acid substitutions relative to SEQ ID NO:7. In still otherembodiments, the amino acid substitutions relative to SEQ ID NO:7 occurat a position within amino acid residues 632-663 of SEQ ID NO:7. In yetother embodiments, the therapeutic protein comprises an amino acidsequence which is not naturally occurring.

In some embodiments, the disclosure teaches an antibody or fragmentthereof which specifically binds the therapeutic protein comprising SEQID NO:3 or a variant thereof. In other embodiments, the antibody orfragment thereof does not bind a protein comprising an amino acidsequence identical to SEQ ID NO:3. In still other embodiments, theantibody or fragment thereof binds a protein comprising an amino acidsequence identical to SEQ ID NO:3 but does not bind a protein comprisingan amino acid sequence identical to SEQ ID NO:1 or SEQ ID NO:7.

In some embodiments, the therapeutic protein increases the barrierfunction of an epithelial cell layer in an in vitro assay, wherein theincrease is relative to the barrier function in the assay in the absenceof the protein. In other embodiments, the in vitro assay is atransepithelial electrical resistance (TEER) assay. In still otherembodiments, the increase in barrier function is an increase inelectrical resistance of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% greater than the electrical resistance in the assay in theabsence of the protein. In some embodiments, the epithelial cell layeris an intestinal epithelial cell layer. In still other embodiments, theintestinal epithelial cell layer is a cell layer which comprisesenterocytes and goblet cells.

In some embodiments, the therapeutic protein decreases the secretion ofa pro-inflammatory cytokine from a cell in an in vitro assay. In otherembodiments, the in vitro assay comprises incubation of monocytic cellswith heat killed E. coli in the presence and absence of the protein. Instill other embodiments, the at least one pro-inflammatory cytokine isselected from the group consisting of TNF-α, IL-17, IL-1β, IL-2, IFN-γ,IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3α, CXCL1, and IL-23.

In some embodiments, the therapeutic protein increases secretion of ananti-inflammatory cytokine from a cell in an in vitro assay. In otherembodiments, the in vitro assay comprises incubation of a monocyte withheat killed E. coli in the presence and absence of the protein. In stillembodiments, the at least one anti-inflammatory cytokine is selectedfrom the group consisting of IL-4, IL-10, IL-13, IFN-α, and TGF-β.

In some embodiments, the therapeutic protein reduces intestinal tissuepathology in a subject administered the protein. In some embodiments,the subject was induced to have intestinal tissue damage by treatmentwith a chemical. In other embodiments, the subject was treated with thechemical dextran sodium sulfate (DSS) to induce intestinal tissuedamage. In still other embodiments, the subject is a mammal. In yetother embodiments, the animal is a rodent. In other embodiments, thesubject is a non human primate.

In some embodiments, the therapeutic protein reduces gastrointestinalinflammation in a subject administered the protein. In otherembodiments, the therapeutic protein reduces intestinal mucosainflammation in the subject. In still other embodiments, the therapeuticprotein improves intestinal epithelial cell barrier function orintegrity in the subject.

In some embodiments, the therapeutic protein increases the amount ofmucin in intestinal tissue in a subject administered said protein.

In some embodiments, the therapeutic protein increases intestinalepithelial cell wound healing in a subject administered the protein. Inother embodiments, the therapeutic protein increases intestinalepithelial cell wound healing in an in vitro assay.

In some embodiments, the therapeutic protein prevents or reduces colonshortening in a subject administered the protein.

In some embodiments, the therapeutic protein modulates (i.e. increasesor decreases) a cytokine in the blood, plasma, serum, tissue and/ormucosa of a subject administered the protein.

In some embodiments, the therapeutic protein decreases the levels of atleast one pro-inflammatory cytokine in the blood, plasma, serum, tissueand/or mucosa of the subject. In other embodiments, the at least onepro-inflammatory cytokine is selected from the group consisting ofTNF-α, IL-17, IL-1β, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8,MCP-1, MIP-3α, CXCL1, and IL-23.

In some embodiments, the therapeutic protein increases the levels of atleast one anti-inflammatory cytokine in the blood, plasma, serum, tissueand/or mucosa of the subject. In other embodiments, the at least oneanti-inflammatory cytokine is selected from the group consisting ofIL-4, IL-10, IL-13, IFN-α, and TGF-β.

In some embodiments, the therapeutic protein decreases the level of atleast one anti-inflammatory cytokine in the blood, plasma, serum, tissueand/or mucosa of the subject. In other embodiments, the at least oneanti-inflammatory cytokine is selected from the group consisting ofIL-4, IL-10, IL-13, IFN-α, and TGF-β.

In some embodiments, the disclosure teaches polynucleotides encoding anovel protein therapeutic and methods of expressing said nucleic acidsin a host cell. In a particular embodiment, the polynucleotide comprisesa sequence which encodes a therapeutic protein that is at least about70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9%, or 100% identical to SEQ ID NO:3. In other embodiments, thepolynucleotide comprises a sequence which encodes a protein that is atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% identical to SEQ IDNO:3 and less than 100% identical to SEQ ID NO:3. In still otherembodiments, the polynucleotide encodes a protein which is anon-naturally occurring variant of SEQ ID NO:1 or SEQ ID NO:7. In stillother embodiments, the polynucleotide is codon-optimized for expressionin a recombinant host cell. In yet other embodiments, the polynucleotideis codon-optimized for expression in E. coli.

In some embodiments, the disclosure teaches a nucleic acid whichcomprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO:4. In other embodiments, the nucleic acid comprises a sequence whichis at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO:4 and less than 100%identical to SEQ ID NO:4. In other embodiments, the nucleic acidcomprises a sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO:8 and less than 100% identical to SEQ ID NO:8. In yet otherembodiments, the nucleic acid comprises a sequence which is anon-naturally occurring variant of SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:8.

In some embodiments, the therapeutic protein is chemically modified atthe N-terminus and/or the C-terminus. In other embodiments, theN-terminus of the protein is chemically modified by acetylation. Instill other embodiments, the C-terminus is chemically modified byamidation.

In some embodiments, the therapeutic protein is pegylated.

In some embodiments, the therapeutic protein is substantially purifiedand which is modified by glycosylation, ubiquitination, nitrosylation,methylation, acetylation, or lipidation.

In some embodiments, the therapeutic protein is fused to second protein.In other embodiments, the second protein is not the therapeutic protein.In still other embodiments, the second protein is an immunoglobulin Fcdomain or a human serum albumin protein domain.

In some aspects, the disclosure provides a pharmaceutical compositionfor treating an inflammatory bowel disease, comprising: a therapeuticprotein comprising an amino acid sequence having at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%,99.7%, 99.8% or 100% sequence identity to SEQ ID NO:3 and apharmaceutically acceptable carrier. In some embodiments, thetherapeutic protein is purified or substantially purified. In someembodiments, the protein comprises the amino acid sequence of SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7. In an alternative embodiment, theprotein does not comprise a sequence which is identical to SEQ ID NO:3or the protein is a non-naturally occurring variant of SEQ ID NO:3 orSEQ ID NO:7.

In some embodiments, the pharmaceutical composition is formulated forrectal, parenteral, intravenous, topical, oral, dermal, transdermal, orsubcutaneous administration. In other embodiments, the pharmaceuticalcomposition is a liquid, a gel, or a cream. In still other embodiments,the pharmaceutical composition is a solid composition comprising anenteric coating.

In some embodiments, the pharmaceutical composition is a cream, acapsule, a liquid, a gel, or an emulsion.

In some embodiments, the pharmaceutical composition is formulated toprovide delayed release. In other embodiments, the delayed release isrelease into the gastrointestinal tract. In yet other embodiments, thedelayed release is into the mouth, the small intestine, the largeintestine and/or the rectum.

In some embodiments, the pharmaceutical composition is formulated toprovide sustained release. In other embodiments, the sustained releaseis release into the gastrointestinal tract. In yet other embodiments,the sustained release is into the mouth, the small intestine, the largeintestine and/or the rectum. In still other embodiments, the sustainedrelease composition releases the therapeutic formulation over a timeperiod of about 1 to 20 hours, 1 to 10 hours, 1 to 8 hours, 4 to 12hours or 5 to 15 hours.

In some embodiments, the pharmaceutical composition further comprises asecond therapeutic agent. In other embodiments, the second therapeuticagent is selected from the group consisting of an anti-diarrheal, a5-aminosalicylic acid compound, an anti-inflammatory agent, anantibiotic, an anti-cytokine agent, an anti-inflammatory cytokine agent,a steroid, a corticosteroid, an immunosuppressant, a JAK inhibitor, ananti-integrin biologic, an anti-IL12/23R biologic, and a vitamin.

In some embodiments, the pharmaceutical composition further comprises aprotease inhibitor. In still other embodiments, the protease inhibitorinhibits degradation of the therapeutic protein in the presence of fecalmatter and/or in the presence of blood.

As aforementioned, these novel protein therapeutics are able to promoteepithelial barrier function and integrity in a subject. In someembodiments, the epithelial barrier function is intestinal epithelialbarrier function. Additionally, the therapeutic effect of the proteinsincludes suppression of an inflammatory immune response in an IBDindividual. Thus, the disclosure provides detailed guidance for methodsof utilizing the taught therapeutic proteins to treat a host ofgastrointestinal inflammatory conditions, and disease states involvingcompromised gastrointestinal epithelial barrier integrity.

In some embodiments, a method for treating an inflammatory bowel diseaseor disorder in a patient in thereof is provided, comprising:administering to the patient a pharmaceutical composition, comprising:i) a therapeutic protein comprising an amino acid sequence having atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9%, or 100% sequence identity to SEQ ID NO:3, SEQ ID NO:5 or SEQID NO:7; and ii) a pharmaceutically acceptable carrier. In otherembodiments of the method, the protein comprises an amino acid sequenceidentical to SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. In still otherembodiments, the protein is not identical in sequence or in length toSEQ ID NO:7 or is a non-naturally occurring variant of SEQ ID NO:7.

In some embodiments, the patient has been diagnosed with intestinalinflammation. In other embodiments, the intestinal inflammation is inthe small intestine and/or the large intestine. In still otherembodiments, the intestinal inflammation is in the rectum. In stillother embodiments, the patient has been diagnosed with pouchitis.

In some embodiments, the patient has been diagnosed with intestinalulcers. In other embodiments, the patient has been diagnosed withdraining enterocutaneous and/or rectovaginal fistulas.

In some embodiments, the patient has been diagnosed with Crohn's disease(CD). In other embodiments, the CD is mildly active CD. In still otherembodiments, the CD is moderately to severely active CD. In yet otherembodiments, the patient has been diagnosed with pediatric CD.

In some embodiments, the patient has been diagnosed with short bowelsyndrome or irritable bowel syndrome.

In some embodiments, the patient has been diagnosed with mucositis. Inother embodiments, the mucositis is oral mucositis. In still otherembodiments, the mucositis is chemotherapy-induced mucositis, radiationtherapy-induced mucositis, chemotherapy-induced oral mucositis, orradiation therapy-induced oral mucositis. In yet other embodiments, themucositis is gastrointestinal mucositis. In still other embodiments, thegastrointestinal mucositis is mucositis of the small intestine, thelarge intestine, or the rectum.

In some embodiments, the administering to a patient diagnosed with CDresulted in a reduced number of draining enterocutaneous and/orrectovaginal fistulas. In other embodiments, the administering maintainsfistula closure in adult patients with fistulizing disease.

In other embodiments, the patient has been diagnosed with ulcerativecolitis (UC). In other embodiments, the UC is mildly active UC. In stillother embodiments, the UC is moderately to severely active UC. In stillother embodiments, the patient has been diagnosed with pediatric UC.

In some embodiments, the patient is in clinical remission from an IBD.In other embodiments, the patient is in clinical remission from UC,pediatric UC, CD or pediatric CD.

In some embodiments, the patient has an inflammatory bowel disease ordisorder other than Crohn's disease or ulcerative colitis. In otherembodiments, the patient has at least one symptom associated withinflammatory bowel disease.

In some embodiments, the administering reduces gastrointestinalinflammation and/or reduces intestinal mucosa inflammation associatedwith inflammatory bowel disease in the patient. In other embodiments,the administering improves intestinal epithelial cell barrier functionor integrity in the patient.

In some embodiments, after the administering the patient experiences areduction in at least one symptom associated with an inflammatory boweldisease or disorder. In other embodiments, the at least one symptom isselected from the group consisting of abdominal pain, blood in stool,pus in stool, fever, weight loss, frequent diarrhea, fatigue, reducedappetite, nausea, cramps, anemia, tenesmus, and rectal bleeding. Instill other embodiments, after the administering the patient experiencesreduced frequency of diarrhea, reduced blood in stool and/or reducedrectal bleeding.

In some embodiments, the patient has experienced inadequate response toconventional therapy. In other embodiments, the conventional therapy istreatment with an aminosalicylate, a corticosteroid, a thiopurine,methotrexate, a JAK inhibitor, a sphingosine 1-phosphate (SiP) receptorinhibitor, an anti-integrin biologic, an anti-IL12/23R or anti-IL23/p10biologic, and/or an anti-tumor necrosis factor agent or biologic.

In some embodiments, the administering modulates (i.e. increases ordecreases) levels of a cytokine in the blood, plasma, serum, mucosa ortissue of the patient.

In some embodiments, the administering suppresses the levels of at leastone pro-inflammatory cytokine in the blood, plasma, serum, mucosa ortissue of the patient. In other embodiments, the at least onepro-inflammatory cytokine is selected from the group consisting ofTNF-α, IL-17, IL-1β, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8,MCP-1, MIP-3α, CXCL1, and IL-23.

In some embodiments, the administering increases the levels of at leastone anti-inflammatory cytokine in the blood, plasma, serum, mucosa ortissue of the patient. In other embodiments, the at least oneanti-inflammatory cytokine is selected from the group consisting ofIL-4, IL-10, IL-13, IFN-α, and TGF-β.

In some embodiments, the administering decreases the level of at leastone anti-inflammatory cytokine in the blood, plasma, serum, mucosa ortissue of the patient. In other embodiments, the at least oneanti-inflammatory cytokine is selected from the group consisting ofIL-4, IL-10, IL-13, IFN-α, and TGF-β.

In some embodiments, the administering increases the amount of mucin inintestinal lumen of the patient.

In some embodiments, the administering increases intestinal epithelialcell wound healing in the patient.

In some embodiments, the administering prevents or reduces colonshortening in the patient.

In some embodiments, the administering comprises rectal, intravenous,parenteral, oral, topical, dermal, transdermal or subcutaneousadministering of the pharmaceutical composition to the patient. In otherembodiments, the administering is to the gastrointestinal lumen.

In some embodiments, the patient is also administered at least onesecond therapeutic agent. In other embodiments, the at least one secondtherapeutic agent is selected from the group consisting of ananti-diarrheal, an anti-inflammatory agent, an antibody, an antibiotic,or an immunosuppressant. In still other embodiments, the at least onesecond therapeutic agent is an aminosalicylate, a steroid, or acorticosteroid. In other embodiments, the at least one secondtherapeutic agent is selected from the group consisting of adalimumab,pegol, golimumab, infliximab, vedolizumab, ustekinumab, tofacitinib, andcertolizumab or certolizumab pegol.

In some aspects, an expression vector is provided, comprising anexogenous polynucleotide that encodes a therapeutic protein comprisingan amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity toSEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. In other embodiments, thepolynucleotide encodes a protein comprising the amino acid sequence ofSEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. In still other embodiments, thepolynucleotide encodes a protein comprising an amino acid sequence thatis not identical to SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

In some aspects, an expression system is provided, comprising a hostcell and the expression vector comprising the aforementioned exogenouspolynucleotide.

In some embodiments, the host cell is prokaryotic or eukaryotic. Inother embodiments, the host cell is mammalian cell, a yeast cell or abacterial cell. In still other embodiments, the bacterial cell isEscherichia coli. In yet other embodiments, the mammalian cell is a CHOcell.

In some aspects, a method of producing the therapeutic protein isprovided.

In some embodiments, the method for producing the therapeutic proteincomprises transforming or transfecting the aforementioned host cell withthe aforementioned expression vector, culturing the transformed ortransfected host cell under conditions sufficient for the expression ofthe aforementioned protein encoded by the aforementioned exogenouspolynucleotide. In other embodiments, the method further comprisespurifying the protein from the transformed or transfected host cell andculture media.

In some embodiments, methods of treating a disease—such as an intestinalepithelium barrier function associated disease—are provided, whichutilize any sequence disclosed in the current application and sequencelisting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show restoration, by SG-14, of epithelial barrierintegrity following inflammation induced disruption, as described inExample 2.

FIG. 2A shows effects of SG-14 administration on TNF-α productioninduced by heat killed Escherichia coli (HK E. coli), as described inExample 3.

FIG. 2B shows effects of SG-14 administration on IL-23 productioninduced by HK E. coli, as described in Example 3.

FIG. 3 shows effects of SG-14 administration on IL-10 production inducedby HK E. coli, as described in Example 4.

FIG. 4 shows effects of SG-14 administration on mucin expressionfollowing stimulation with HK E. coli, as described in Example 5.

FIG. 5 effects of SG-14 administration on epithelial cell wound healing,as described in Example 6.

FIG. 6 shows effects of SG-14 administration on epithelial centricbarrier function readouts in a DSS model of inflammatory bowel disease,as described in Example 7.

FIG. 7 effects of SG-14 administration on inflammatory readoutsresponsive to impaired barrier function in a DSS model of inflammatorybowel disease, as described in Example 7.

FIG. 8 shows effects of SG-14 administration on body weight loss in aDSS model of inflammatory bowel disease, as described in Example 7.

FIG. 9 shows effects of SG-14 administration on gross pathology in a DSSmodel of inflammatory bowel disease, as described in Example 7.

FIG. 10 shows effects of SG-14 administration on colon length in a DSSmodel of inflammatory bowel disease, as described in Example 7.

FIG. 11 shows effects of SG-14 administration on colon weight to lengthratio in a DSS model of inflammatory bowel disease, as described inExample 7.

FIG. 12A and FIG. 12B show effects of SG-14 administration on colonictissue pathology in a DSS model of inflammatory bowel disease, asdescribed in Example 7.

DETAILED DESCRIPTION

The present disclosure provides novel protein therapeutics that areuseful in the treatment of subjects suffering from symptoms associatedwith gastrointestinal disorders. For example, these proteins can promoteor enhance epithelial barrier function and/or integrity. The protein mayalso suppress the inflammatory immune response in an IBD individual. Theprotein therapeutic provided herein is useful in treating the numerousdiseases that are associated with decreased gastrointestinal epithelialcell barrier function or integrity and inflammation of the intestine.

In the present disclosure, provided are also protein variants that havetherapeutic activity comparable to or superior to the original protein,but the protein variants have enhanced stability through themanufacturing and processing of the protein therapeutic products as wellas under long-term storage conditions.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, molecular biology, celland cancer biology, immunology, microbiology, pharmacology, and proteinand nucleic acid chemistry, described herein, are those well-known andcommonly used in the art. Thus, while the following terms are believedto be well understood by one of ordinary skill in the art, the followingdefinitions are set forth to facilitate explanation of the presentlydisclosed subject matter.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated component, or group of components, but not the exclusion of anyother components, or group of components.

The term “a” or “an” refers to one or more of that entity, i.e. canrefer to a plural referents. As such, the terms “a” or “an,” “one ormore,” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

The terms “gastrointestinal” or “gastrointestinal tract,” “alimentarycanal,” and “intestine,” as used herein, may be used interchangeably torefer to the series of hollow organs extending from the mouth to theanus and including the mouth, esophagus, stomach, small intestine, largeintestine, rectum and anus. The terms “gastrointestinal” or“gastrointestinal tract,” “alimentary canal,” and “intestine” are notalways intended to be limited to a particular portion of the alimentarycanal.

The term “SG-14” as used herein refers to a protein comprising the aminoacid sequence of SEQ ID NO:3 and also to variants thereof having thesame or similar functional activity as described herein. Accordingly,SG-14 can refer herein to proteins comprising or consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 or variants or fragmentsthereof. In U.S. Provisional Patent Application No. (62/508,501, filedMay 19, 2017, to which the present specification claims priority andwhich is incorporated herein by reference in its entirety) the term“Experimental Protein 3” and variants thereof was used and is synonymouswith SG-14 as used herein or variants thereof.

A “signal sequence” (also termed “presequence,” “signal peptide,”“leader sequence,” or “leader peptide”) refers to a sequence of aminoacids located at the N-terminus of a nascent protein, and which canfacilitate the secretion of the protein from the cell. The resultantmature form of the extracellular protein lacks the signal sequence,which is cleaved off during the secretion process.

The recitations “sequence identity,” “percent identity,” “percenthomology,” or for example, comprising a “sequence 50% identical to,” asused herein, refer to the extent that sequences are identical on anucleotide-by-nucleotide or amino acid-by-amino acid basis, over awindow of comparison. Thus, a “percentage of sequence identity” may becalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, I) or the identical aminoacid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr,Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity.

Calculations of sequence similarity or sequence identity betweensequences (the terms are used interchangeably herein) can be performedas follows. To determine the percent identity of two amino acidsequences, or of two nucleic acid sequences, the sequences can bealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In certain embodiments, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, 60%, and evenmore preferably at least 70%, 80%, 90%, or 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The phrases “substantially similar” and “substantially identical” in thecontext of at least two nucleic acids or polypeptides typically meansthat a polynucleotide or polypeptide comprises a sequence that has atleast about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7% or even 99.8% sequenceidentity, in comparison with a reference polynucleotide or polypeptide.In some embodiments, substantially identical polypeptides differ only byone or more conservative amino acid substitutions. In some embodiments,substantially identical polypeptides are immunologically cross-reactive.In some embodiments, substantially identical nucleic acid moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

Related (and derivative) proteins encompass “variant” proteins. Variantproteins can differ from another (i.e., parental) protein and/or fromone another by a small number of amino acid residues. A variant mayinclude one or more amino acid mutations (e.g., amino acid deletion,insertion or substitution) as compared to the parental protein fromwhich it is derived.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, a conservatively modified variant refers to nucleic acidsencoding identical amino acid sequences, or amino acid sequences thathave one or more “conservative substitutions.” An example of aconservative substitution is the exchange of an amino acid in one of thefollowing groups for another amino acid of the same group (see U.S. Pat.No. 5,767,063; Kyte and Doolittle (1982) J. Mol. Biol. 157:105-132). (1)Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, Met; (2) Neutralhydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basic: Asn, Gln,His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro;(6) Aromatic: Trp, Tyr, Phe; and (7) Small amino acids: Gly, Ala, Ser.Thus, the term “conservative substitution” with respect to an amino aciddenotes that one or more amino acids are replaced by another, chemicallysimilar residue, wherein said substitution does not generally affect thefunctional properties of the protein. Examples include substitution ofamino acid residues with similar characteristics, e.g., small aminoacids, acidic amino acids, polar amino acids, basic amino acids,hydrophobic amino acids and aromatic amino acids. In some embodiments,the disclosure provides for proteins that have at least onenon-naturally occurring, conservative amino acid substitution relativeto the amino acid sequence identified in SEQ ID NO:3 or SEQ ID NO:5 orSEQ ID NO:7. Some common exemplary examples of conservative amino acidsubstitutions are found below.

The term “amino acid” or “any amino acid” refers to any and all aminoacids, including naturally occurring amino acids (e.g., α-amino acids),unnatural amino acids, modified amino acids, and unnatural ornon-natural amino acids. It includes both D- and L-amino acids. Naturalamino acids include those found in nature, such as, e.g., the 23 aminoacids that combine into peptide chains to form the building-blocks of avast array of proteins. These are primarily L stereoisomers, although afew D-amino acids occur, e.g., in bacterial envelopes and someantibiotics. The 20 “standard,” natural amino acids are listed in theabove tables. The “non-standard,” natural amino acids are pyrrolysine(found in methanogenic organisms and other eukaryotes), selenocysteine(present in many noneukaryotes as well as most eukaryotes), andN-formylmethionine (encoded by the start codon AUG in bacteria,mitochondria and chloroplasts). “Unnatural” or “non-natural” amino acidsare non-proteinogenic amino acids (i.e., those not naturally encoded orfound in the genetic code) that either occur naturally or are chemicallysynthesized. Over 140 unnatural amino acids are known and thousands ofmore combinations are possible. “Modified” amino acids include aminoacids (e.g., natural amino acids) that have been chemically modified toinclude a group, groups, or chemical moiety not naturally present on theamino acid.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature, or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence. As used herein, a “synthetic amino acid sequence” or“synthetic peptide sequence” or “synthetic polypeptide sequence” or“synthetic protein sequence” is an amino acid sequence that is not knownto occur in nature, or that is not naturally occurring. Generally, sucha synthetic amino acid sequence will comprise at least one amino aciddifference when compared to any other naturally occurring amino acidsequence.

As used herein, a “synthetic protein” or “synthetic therapeutic protein”means a protein that comprises an amino acid sequence that contains oneor more amino acids substituted with different amino acids relative to anaturally occurring amino acid sequence. That is, a “synthetic protein”comprises an amino acid sequence that has been altered to contain atleast one non-naturally occurring substitution modification at a givenamino acid position(s) relative to a naturally occurring amino acidsequence.

The term “about” as used herein with respect to % sequence identity, or% sequence homology, of a nucleic acid sequence, or amino acid sequence,means up to and including f1.0% in 0.1% increments. For example, “about90%” sequence identity includes 89.0%, 89.1%, 89.2%, 89.3%, 89.4%,89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%, 90.3%, 90.4%,90.5%, 90.6%, 90.7%, 90.8%, 90.9%, and 91%. If not used in the contextof % sequence identity, then “about” means±1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, or 10%, depending upon context of the value in question.

For the most part, the names of natural and non-natural aminoacylresidues used herein follow the naming conventions suggested by theIUPAC Commission on the Nomenclature of Organic Chemistry and theIUPAC-IUB Commission on Biochemical Nomenclature as set out in“Nomenclature of α-Amino Acids (Recommendations, 1974)” Biochemistry,14(2), (1975). To the extent that the names and abbreviations of aminoacids and aminoacyl residues employed in this specification and appendedclaims differ from those suggestions, they will be made clear to thereader.

Throughout the present specification, unless natural amino acids arereferred to by their full name (e.g., alanine, arginine, etc.), they aredesignated by their conventional three-letter or single-letterabbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.).Unless otherwise indicated, three-letter and single-letter abbreviationsof amino acids refer to the L-isomeric form of the amino acid. The term“L-amino acid,” as used herein, refers to the “L” isomeric form of apeptide, and conversely the term “D-amino acid” refers to the “D”isomeric form of a peptide (e.g., Dasp, (D)Asp or D-Asp; Dphe, (D)Phe orD-Phe). Amino acid residues in the D isomeric form can be substitutedfor any L-amino acid residue, as long as the desired function isretained by the peptide. D-amino acids may be indicated as customary inlower case when referred to using single-letter abbreviations.

In the case of less common or non-natural amino acids, unless they arereferred to by their full name (e.g., sarcosine, omithine, etc.), three-or four-character codes are frequently employed for residues thereof,including, Sar or Sarc (sarcosine, i.e. N-methylglycine), Aib(α-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa(2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), Gaba(γ-aminobutanoic acid), β-Pro (pyrrolidine-3-carboxylic acid), and 8Ado(8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), βhPro(β-homoproline), βhPhe (β-homophenylalanine) and Bip (β,βdiphenylalanine), and Ida (Iminodiacetic acid).

Among sequences disclosed herein are sequences incorporating a “Hy-”moiety at the amino terminus (N-terminus) of the sequence, and either an“—OH” moiety or an “—NH2” moiety at the carboxy terminus (C-terminus) ofthe sequence. In such cases, and unless otherwise indicated, a “Hy-”moiety at the N-terminus of the sequence in question indicates ahydrogen atom, corresponding to the presence of a free primary orsecondary amino group at the N-terminus, while an “—OH” or an “—NH2”moiety at the C-terminus of the sequence indicates a hydroxy group or anamino group, corresponding to the presence of an amido (CONH2) group atthe C-terminus, respectively. In each sequence of the disclosure, aC-terminal “—OH” moiety may be substituted for a C-terminal “—NH2”moiety, and vice-versa.

The term “Ac,” as used herein, refers to acetyl protection throughacylation of the C- or N-terminus of a polypeptide. In certain peptidesshown herein, the NH2 locates at the C-terminus of the peptide indicatesan amino group. The term “carboxy,” as used herein, refers to —CO₂H.

The term “pharmaceutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the peptides, proteins, or compounds ofthe present disclosure, which are water or oil-soluble or dispersible,which are suitable for treatment of diseases without undue toxicity,irritation, and allergic response; which are commensurate with areasonable benefit/risk ratio, and which are effective for theirintended use. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting an amino groupwith a suitable acid. Representative acid addition salts includeacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,formate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isethionate), lactate, maleate,mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, picrate, pivalate, propionate, succinate, tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate, and undecanoate. Also, amino groups in thecompounds of the present disclosure can be quaternized with methyl,ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl,diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, andsteryl chlorides, bromides, and iodides; and benzyl and phenethylbromides. Examples of acids which can be employed to formtherapeutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric. A pharmaceuticallyacceptable salt may suitably be a salt chosen, e.g., among acid additionsalts and basic salts. Examples of acid addition salts include chloridesalts, citrate salts and acetate salts. Examples of basic salts includesalts where the cation is selected among alkali metal cations, such assodium or potassium ions, alkaline earth metal cations, such as calciumor magnesium ions, as well as substituted ammonium ions. Other examplesof pharmaceutically acceptable salts are described in “Remington'sPharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, PA, USA, 1985 (and more recent editionsthereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rdedition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA,2007, and in J. Pharm. Sci. 66: 2 (1977). Also, for a review on suitablesalts, see Handbook of Pharmaceutical Salts: Properties. Selection, andUse by Stahl and Wermuth (Wiley-VCH, 2002).

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to a target polynucleotide.

As used herein, the phrases “recombinant construct,” “expressionconstruct,” “chimeric construct,” “construct,” and “recombinant DNAconstruct” are used interchangeably herein and are well-known to theordinarily skilled artisan.

As used herein, the term “host cell” refers to a cell or cell line intowhich a recombinant expression vector for production of a polypeptidemay be introduced for expression of the polypeptide.

The terms “isolated,” “purified,” “separated,” and “recovered” as usedherein refer to a material (e.g., a protein, nucleic acid, or cell) thatis removed from at least one component with which it is naturallyassociated, for example, at a concentration of at least 90% by weight,or at least 95% by weight, or at least 98% by weight of the sample inwhich it is contained. For example, these terms may refer to a materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state, such as, forexample, an intact biological system.

The terms “patient,” “subject,” and “individual” may be usedinterchangeably and refer to either a human or a non-human animal. Theseterms include mammals such as humans, non-human primates, livestockanimals (e.g., bovines, porcines), companion animals (e.g., canines,felines) and rodents (e.g., mice and rats). In certain embodiments, theterms refer to a human patient. In exemplary embodiments, the termsrefer to a human patient that suffers from a gastrointestinalinflammatory condition.

As used herein, “improved” should be taken broadly to encompassimprovement in an identified characteristic of a disease state, saidcharacteristic being regarded by one of skill in the art to generallycorrelate, or be indicative of, the disease in question, as compared toa control, or as compared to a known average quantity associated withthe characteristic in question. For example, “improved” epithelialbarrier function associated with application of a protein of thedisclosure can be demonstrated by comparing the epithelial barrierintegrity of a human treated with a protein of the disclosure, ascompared to the epithelial barrier integrity of a human not treated.Alternatively, one could compare the epithelial barrier integrity of ahuman treated with a protein of the disclosure to the average epithelialbarrier integrity of a human, as represented in scientific or medicalpublications known to those of skill in the art. In the presentdisclosure, “improved” does not necessarily demand that the data bestatistically significant (i.e. p<0.05); rather, any quantifiabledifference demonstrating that one value (e.g. the average treatmentvalue) is different from another (e.g. the average control value) canrise to the level of “improved.”

As used herein, “inhibiting and suppressing” and like terms should notbe construed to require complete inhibition or suppression, althoughthis may be desired in some embodiments. Thus, an “inhibited immuneresponse” or the “inhibition of inflammatory cytokines” does not requireabsolute inhibition.

Thus, as used herein, the terms “increase,” “suppress” or “reduce,” orgrammatical equivalents thereof, indicate values that are relative to areference (e.g., baseline) measurement, such as a measurement takenunder comparable conditions (e.g., in the same individual prior toinitiation of treatment described herein, or a measurement in a controlindividual (or multiple control individuals) in the absence oftreatment) described herein. In some embodiments, a suitable control isa baseline measurement, such as a measurement in the same individualprior to initiation of the treatment described herein, or a measurementin a control individual (or multiple control individuals) in the absenceof the treatment described herein.

As used herein, the term “IBD” or “inflammatory bowel disease” refers toconditions in which individuals have chronic or recurring immuneresponse and inflammation of the gastrointestinal (GI) tract. The twomost common inflammatory bowel diseases are ulcerative colitis (UC) andCrohn's disease (CD).

As used herein, the term “therapeutically effective amount” refers to anamount of a therapeutic agent (e.g., a peptide, polypeptide, or proteinof the disclosure), which confers a therapeutic effect on the treatedsubject, at a reasonable benefit/risk ratio applicable to any medicaltreatment. Such a therapeutic effect may be objective (i.e., measurableby some test or marker) or subjective (i.e., subject gives an indicationof, or feels an effect). In some embodiments, “therapeutically effectiveamount” refers to an amount of a therapeutic agent or compositioneffective to treat, ameliorate, or prevent (e.g., delay onset of) arelevant disease or condition, and/or to exhibit a detectabletherapeutic or preventative effect, such as by ameliorating symptomsassociated with the disease, preventing or delaying onset of thedisease, and/or also lessening severity or frequency of symptoms of thedisease. A therapeutically effective amount is commonly administered ina dosing regimen that may comprise multiple unit doses. For anyparticular therapeutic agent, a therapeutically effective amount (and/oran appropriate unit dose within an effective dosing regimen) may vary,for example, depending on route of administration, or on combinationwith other therapeutic agents. Alternatively or additionally, a specifictherapeutically effective amount (and/or unit dose) for any particularpatient may depend upon a variety of factors including the particularform of disease being treated; the severity of the condition orpre-condition; the activity of the specific therapeutic agent employed;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration, route ofadministration, and/or rate of excretion or metabolism of the specifictherapeutic agent employed; the duration of the treatment; and likefactors as is well known in the medical arts. The current disclosureutilizes therapeutically effective amounts of novel proteins, andcompositions comprising same, to treat a variety of diseases, such as:gastrointestinal inflammatory diseases or diseases involvinggastrointestinal epithelial barrier malfunction. The therapeuticallyeffective amounts of the administered protein, or compositionscomprising same, will in some embodiments reduce inflammation associatedwith IBD or repair gastrointestinal epithelial barrier integrity and/orfunction.

As used herein, the term “treatment” (also “treat” or “treating”) refersto any administration of a therapeutic agent (e.g., a peptide,polypeptide, or protein of the disclosure), according to a therapeuticregimen that achieves a desired effect in that it partially orcompletely alleviates, ameliorates, relieves, inhibits, delays onset of,reduces severity of and/or reduces incidence of one or more symptoms orfeatures of a particular disease, disorder, and/or condition (e.g.,chronic or recurring immune response and inflammation of thegastrointestinal (GI) tract); in some embodiments, administration of thetherapeutic agent according to the therapeutic regimen is correlatedwith achievement of the desired effect. Such treatment may be of asubject who does not exhibit signs of the relevant disease, disorderand/or condition and/or of a subject who exhibits only early signs ofthe disease, disorder, and/or condition. Alternatively or additionally,such treatment may be of a subject who exhibits one or more establishedsigns of the relevant disease, disorder and/or condition. In someembodiments, treatment may be of a subject who has been diagnosed assuffering from the relevant disease, disorder, and/or condition. In someembodiments, treatment may be of a subject known to have one or moresusceptibility factors that are statistically correlated with increasedrisk of development of the relevant disease, disorder, and/or condition.

“Pharmaceutical” implies that a composition, reagent, method, and thelike, are capable of a pharmaceutical effect, and also that thecomposition is capable of being administered to a subject safely.“Pharmaceutical effect,” without limitation, can imply that thecomposition, reagent, or method, is capable of stimulating a desiredbiochemical, genetic, cellular, physiological, or clinical effect, in atleast one individual, such as a mammalian subject, for example, a human,in at least 5% of a population of subjects, in at least 10%, in at least20%, in at least 30%, in at least 50% of subjects, and the like.“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for safe use in animals, andmore particularly safe use in humans. “Pharmaceutically acceptablevehicle” or “pharmaceutically acceptable excipient” refers to a diluent,adjuvant, excipient or carrier with which a protein as described hereinis administered.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a subject that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease, or causing the symptom to develop with lessseverity than in absence of the treatment). “Prevention” or“prophylaxis” may refer to delaying the onset of the disease ordisorder.

The therapeutic pharmaceutical compositions taught herein may compriseone or more natural products. However, in certain embodiments, thetherapeutic pharmaceutical compositions themselves do not occur innature. Further, in certain embodiments, the therapeutic pharmaceuticalcompositions possess markedly different characteristics, as compared toany individual naturally occurring counterpart, or compositioncomponent, which may exist in nature. That is, in certain embodiments,the pharmaceutical compositions taught herein-which comprise atherapeutically effective amount of a purified protein-possess at leastone structural and/or functional property that impart markedly differentcharacteristics to the composition as a whole, as compared to any singleindividual component of the composition as it may exist naturally. Thecourts have determined that compositions comprising natural products,which possess markedly different characteristics as compared to anyindividual component as it may exist naturally, are statutory subjectmatter. Thus, the taught therapeutic pharmaceutical compositions as awhole possess markedly different characteristics. These characteristicsare illustrated in the data and examples taught herein.

Details of the disclosure are set forth herein. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, illustrative methodsand materials are now described. Other features, objects, and advantagesof the disclosure will be apparent from the description and from theclaims.

Therapeutic Proteins Derived from the Microbiome—Overview of theDisclosure

Numerous diseases and disorders are associated with decreasedgastrointestinal epithelial cell barrier function or integrity. Thesediseases and disorders are multifaceted and present diagnostically in amyriad of ways. One such disease is inflammatory bowel disease (IBD),the incidence and prevalence of which is increasing with time and indifferent regions around the world, indicating its emergence as a globaldisease. (Molodecky et al., Gastroenterol 142:46-54, 2012). IBD is acollective term that describes conditions with chronic or recurringimmune response and inflammation of the gastrointestinal (GI) tract. Thetwo most common inflammatory bowel diseases are ulcerative colitis (UC)and Crohn's disease (CD). Both are marked by an abnormal response of theGI immune system. Normally, immune cells protect the body frominfection. In people with IBD, however, this immune system mistakesfood, bacteria, and other materials in the intestine for pathogens andan inflammatory response is launched into the lining of the intestines,creating chronic inflammation. When this happens, the patientexperiences the symptoms of IBD.

IBD involves chronic inflammation of all, or part, of the digestivetract. Both UC and CD usually involve, for example, severe diarrhea,abdominal pain, fatigue, and weight loss. IBD and associated disorderscan be debilitating and sometimes lead to life-threateningcomplications.

With respect to intestinal barrier integrity, loss of integrity of theintestinal epithelium plays a key pathogenic role in IBD. Maloy, KevinJ: Powrie. Fiona. “Intestinal homeostasis and its breakdown ininflammatory bowel disease,” (2011) Nature. 474 (7351): 298-306. It ishypothesized that detrimental changes in the intestinal microbiotainduce an inappropriate or uncontrolled immune response that results indamage to the intestinal epithelium. Breaches in this criticalintestinal epithelium barrier allow further infiltration of microbiotathat, in turn, elicit further immune responses. Thus, IBD is amultifactorial disease that is driven in part by an exaggerated immuneresponse to gut microbiota that can cause defects in epithelial barrierfunction.

Microbiome profiling of IBD patients has revealed distinct profiles suchas increased Proteobacteria, including adherent-invasive E. coli, oftenat the expense of potentially beneficial microbes such as Roseburia spp(Machiels et al., 2014, Cut, 63:1275-1283; Patterson et al., 2017, FrontImmunol, 8:1166; Shawki and McCole, 2017, Cell Mol GastroenterolHepatol, 3:41-50). Moreover, a decrease in Roseburia hominis was linkedwith dysbiosis in patients with ulcerative colitis. IBD affectedindividuals have been found to have 30-50 percent reduced biodiversityof commensal bacteria, such as decreases in Firmicutes (namelyLachnospiraceae) and Bacteroidetes. Further evidence of the role of gutflora in the cause of inflammatory bowel disease is that IBD affectedindividuals are more likely to have been prescribed antibiotics in the2-5 year period before their diagnosis than unaffected individuals. See,Aroniadis O C. Brandt L J, “Fecal microbiota transplantation: past,present and future,” (2013) Curr. Opin. Gastroenterol. 29 (1) (2013):79-84.

Protective bacterial communities, probiotics and bacterially derivedmetabolites have been demonstrated to improve disease in variousclinical and pre-clinical studies. For example, fecal microbial transfer(FMT) experiments have shown some success in IBD patients, althoughchallenges still exist with FMT (Moayyedi et al., 2015,Gastroenterology, 149:102-109 e106; Qazi et al., 2017, Gut Microbes,8:574-588; Narula et al., 2017, Inflamm Bowel Dis, 23:1702-1709). Inother studies treatment with probiotics including VSL #3, Lactobacillusspp. and Bifidobacterium spp. have also shown to have beneficial effectsin humans and animal models (Srutkova et al., 2015, PLoS One,10:e0134050; Pan et al., 2014, Benef Microbes, 5:315-322; Huynh et al.,2009, Inflamm Bowel Dis, 15:760-768; Bibiloni et al., 2005, Am JGastroenterol, 100:1539-1546). Furthermore, bacterial products such asp40 from L. rhamnosus GG and Amuc-1100 from A. muciniphila have beenshown to promote barrier function and protect in animal models of IBDand metabolic disease, receptively (Yan et al., 2011, J Clin Invest,121:2242-2253; Plovier et al., Nat Med, 23:107-113).

While uses of live microbial populations to treat diseases isincreasingly common, such methods rely on the ability of theadministered bacteria to survive in the host or patient and to interactwith the host tissues in a beneficial and therapeutic way. Analternative approach, provided here, is to identify microbially-encodedproteins and variants thereof which can affect cellular functions in thehost and provide therapeutic benefit. Such proteins can be administered,for example, as pharmaceutical compositions comprising a substantiallyisolated or purified therapeutic, bacterially-derived protein or as alive biotherapeutic (bacterium) engineered to express the therapeuticprotein as an exogenous protein. Moreover, methods of treatmentcomprising administration of the therapeutic protein are not limited tothe gut (small intestine, large intestine, rectum) but may also includetreatment of other disorders within the alimentary canal such as oralmucositis.

To identify microbially-derived proteins which may have therapeuticapplication in gastrointestinal inflammatory disorders, mucosal biopsiesfrom humans who were healthy or who were diagnosed with IBD (UC) wereanalyzed to determine the microbial compositions of these mucosalbiopsies. A comparison of the bacterial profiles from healthy vs.diseased subjects identified bacteria that were either likely to bebeneficial (greater numbers in healthy vs. diseased) or detrimental(lower numbers in healthy vs. diseased). Among the bacterial speciesidentified as beneficial was Eubacterium eligens. Eubacterium spp. are agroup of anaerobic Gram-positive nonspore-forming rods and members ofthe bacterial phylum Firmicutes. Extensive bioinformatics analysis wasthen performed to predict proteins encoded by the bacterium and then toidentify those proteins which are likely to be secreted by thebacterium. Proteins which were predicted to be secreted proteins werethen recombinantly expressed and characterized using a series of invitro assays to study the effect of each protein on epithelial barrierintegrity, cytokine production and/or release, and wound healing.Proteins identified as functioning to increase epithelial barrierintegrity were then assessed in an in vivo mouse model for colitis. Onesuch protein, identified herein as “SG-14,” demonstrated both in vitroand in vivo activity indicative of its ability to provide therapeuticbenefit for improving epithelial barrier integrity and for treatingdiseases and disorders associated with epithelial barrier integrity aswell as treating inflammatory gastrointestinal diseases such as IBDs, asdescribed in more detail below.

The SG-14 Protein

The protein referred to herein as SG-14 is encoded by a 1893 nucleotidesequence (SEQ ID NO:4; encoding SEQ ID NO:3) present in the genome ofEubacterium eligens (DSM 3376 Type Strain; also designated C15-B4 as perDSMZ; See, e.g., Holdeman, L. V., Moore, W. E. C. (1974) New genus,Coprococcus, twelve new species, and amended descriptions of fourpreviously described species of bacteria from human feces. Int J SystBacteriol 24, 260-277. Recombinant SG-14 can be expressed with anN-terminal methionine (encoded by the codon ATG) to produce a matureprotein of 148 amino acids (SEQ ID NO:5). A complete genomic sequencefor E. eligens can be found at GenBank accession number CP001104 (thesequence incorporated herein by reference in its entirety). A 16S rRNAgene sequence for the E. eligens DSM 3376 strain can be found at GenBankaccession number NR_074613 (the sequence incorporated herein byreference in its entirety). A similar protein predicted to be encoded bythe E. eligens genomic sequence (GenBank Acc. No. ACR71212, encoded bythe reverse complement of nucleotides 205249-207312 of GenBank Acc. No.CP001104) is 687 amino acids in length (SEQ ID NO:1), wherein residues1-24 (of SEQ ID NO:1) are predicted to be a signal peptide which iscleaved in vivo to produce a mature protein of 663 amino acids (SEQ IDNO:7). Accordingly, it is noted that amino acid residues 1-631 of SEQ IDNO:3 correspond to amino acid residues 25 to 655 of SEQ ID NO:1 Whenrecombinantly expressed, the SG-14 protein will comprise a startmethionine residue such that, e.g., SG-14 comprises or consists of theamino acid sequence of SEQ ID NO:5 (632 amino acids in length), encodedby SEQ ID NO:6 (1896 nucleotides in length) and SEQ ID NO:7 (663 aminoacids in length), encoded by SEQ ID NO:8 (1989 nucleotides in length).

As described in Example 1, SG-14 was recombinantly expressed incommercially available and routinely used expression vectors. Forexample, SG-14 (a protein comprising SEQ ID NO:3), was expressed using apGEX expression vector which expresses the protein of interest with aGST tag and protease site which is cleaved after expression andpurification. This expression system can result in a protein, aftercleavage with a protease, with an N-terminus which differs with respectto a few amino acid residues relative to the wildtype protein (e.g., SEQID NO:3 or SEQ ID NO:5). Moreover, a few exogenous amino acid residuesmay be present at the C-terminus of the protein. Alternative expressionsystems can include but are not limited to, e.g., a pET-28 expressionvector which adds an N-terminal FLAG tag (e.g., DYKDDDDK, SEQ ID NO:9),and a pD451 expression vector which can be used to express the SG-14protein consisting of SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:7 and havingno exogenous N-terminal or C-terminal amino acids. Experiments performedand repeated with these proteins can show that the minor N-terminaland/or C-terminal variations resulting from the use of the differentprotein expression systems and DNA constructs retain equivalentfunctional activity in in vivo and in vitro assays. It is understoodthat unless otherwise indicated, the term “SG-14” refers herein to theamino acid sequence depicted herein as SEQ ID NO:3 and such variants ofthe protein comprising the amino acid sequence of SEQ ID NO:3 (includingbut not limited to SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:7) whereinthe protein comprising SEQ ID NO:3 or a variant thereof possessesessentially the same in vitro and/or in vivo activity as show by assaysdescribed herein as Examples 2-7. SG-14 variants can include variationsin amino acid residues (substitution, insertion, deletion) as well asmodifications such as fusion constructs and post-translationalmodifications (phosphorylation, glycosylation, etc.). Some exemplaryembodiments of the SG-14 protein and encoding nucleic acids are providedin Table 1 below.

TABLE 1 Amino Acid Encoding Nucleic Sequence Acid Sequence SEQ ID NO: 1SEQ ID NO: 2 MIKLNKIKRNCVAAVILTMC ATGATTAAATTAAATAAAATLMTAGCARNSTSTTTASGGE CAAAAGGAATTGCGTAGCCG TTITSAITKEDTDVTHADDACAGTTATTCTTACTATGTGT ENYRVSITGDFTVTSDTSDG CTTATGACTGCTGGCTGTGCVTQSGSVYTITKAGEYTVTG CAGAAATTCAACTTCAACTA LLSEGQLIVDAGNENEVTIVCTACTGCATCAGGCGGTGAG LNGTSITCSSGSPIYVKNAS ACTACCATCACTTCAGCCATEVKIKSEENSFNEVIDNRNE TACTAAAGAAGACACAGATG ATEDSSDDAGNAAIYATCDLTAACACACGCAGATGATGCT KLVGKGALVVTGNYNNGIQS GAGAATTACAGAGTCTCCATKDDLSIKNVIVKVTAVNNAV TACAGGTGATTTCACTGTGA KVNDAVDIESGNIIAISAKGCATCTGATACATCAGATGGA DGIKTSNSSISNKGNQKGIV GTTACACAGTCAGGTTCTGTTITSGNIDIYAACDGIDASY ATACACAATCACAAAGGCTG GADISGDGNLNIYTDTYSEYGTGAATATACAGTAACAGGA SEEVTSSGSSSGTSSGRDSS CTTTTATCAGAAGGACAGCTANKSASANTVSYVATSDTIA TATCGTTGACGCAGGTAATG NAPSGFGGGNMGSGNAPDMSAGAACGAGGTTACTATCGTA NGNAPNMNGSSDRNKTGGNR TTGAACGGAACATCTATCACPGMPGDFNESGNSSGQSYST ATGCTCAAGTGGTTCACCTA KGIKAESEINISGFTINICSTATACGTTAAAAATGCTTCA TDDGIHANSDSGVLETGEDG GAAGTCAAGATTAAATCAGAKGTIVINGGSITISSGDDGM AGAGAACTCATTTAACGAAG HADKQLDVNDGYINVVTSYETAATTGACAATCGTAACGAA GLEAMTINLNGGKIYVYATD GCTACAGAAGATTCTTCTGADGINACTGDGKTSPIVNVTG TGACGCTGGCAACGCAGCAA GYIDVTTTSGDTDGIDSNGNTCTATGCAACATGCGATTTA YVQTGGFVLVKGGSSSGNVS AAACTCGTCGGCAAAGGAGCGSIDVDGTVTITGGTCVALG CTTAGTTGTAACAGGTAATT GVCETPVNSANAYVLGSVSFACAATAATGGTATCCAGAGC SSGSYSLKDSSGNEVISFTV AAGGATGACCTTTCTATTAADGSFSNGWICSDTLTTGSSY AAATGTGATTGTTAAAGTTA TLYRGADSIADWTQESGTMGCTGCTGTGAACAACGCAGTC ASGTGGFGGGNMGGMGGQNG AAAGTCAACGATGCCGTTGA GFGGGRRTATTGAATCTGGAAATATAA TTGCAATCTCCGCTAAAGGC GATGGCATCAAGACTTCTAACAGCAGTATTTCTAACAAGG GCAACCAGAAGGGAATCGTT ACAATCACTAGTGGTAACATTGATATTTATGCAGCCTGTG ACGGTATAGACGCATCTTAC GGGGCAGATATATCAGGTGACGGTAACTTAAACATTTATA CAGATACTTACTCTGAATAT AGTGAAGAAGTTACTTCGTCAGGCAGTTCTTCAGGCACGT CTTCTGGCCGGGACAGCTCT GCTAACAAGTCTGCTTCTGCCAATACTGTTTCTTATGTGG CAACTTCTGACACTATTGCC AACGCACCTAGCGGCTTTGGTGGTGGCAACATGGGTAGCG GCAATGCTCCAGATATGAGT AACGGCAACGCTCCCAATATGAACGGCAGCTCTGATAGAA ATAAGACCGGTGGCAACCGT CCAGGAATGCCTGGTGACTTTAATGAATCCGGTAATTCTT CTGGACAGTCCTACTCAACT AAGGGTATTAAAGCTGAAAGCGAAATAAATATTTCAGGCT TTACAATTAACATATGTTCA ACAGATGATGGTATCCATGCCAACTCTGACTCAGGTGTAC TTGAAACCGGTGAGGACGGC AAAGGAACTATTGTTATCAACGGCGGTTCAATTACAATTT CTTCTGGCGATGACGGCATG CACGCTGACAAACAGCTTGATGTCAATGACGGTTACATTA ATGTAGTAACTTCATATGAA GGACTTGAGGCTATGACTATCAACTTAAATGGCGGCAAGA TATATGTATACGCTACTGAT GATGGCATTAATGCCTGCACAGGTGATGGAAAGACTTCTC CAATTGTCAATGTAACTGGT GGATATATAGATGTCACAACTACGTCTGGTGATACTGATG GTATTGATTCTAATGGAAAT TACGTACAGACCGGTGGATTTGTATTAGTTAAAGGTGGCA GTTCATCTGGAAATGTATCA GGATCAATTGATGTAGATGGTACCGTAACGATAACCGGTG GAACATGCGTTGCCCTCGGT GGTGTATGCGAAACACCTGTAAACTCTGCTAATGCTTATG TATTAGGTTCCGTATCATTC AGTTCTGGAAGCTATTCACTTAAAGATTCTTCTGGCAACG AAGTTATAAGCTTCACTGTT GACGGTTCATTTAGCAACGGCTGGATATGTTCTGACACTC TTACAACCGGCTCAAGCTAC ACACTCTACCGGGGGGCAGACTCTATTGCAGACTGGACTC AGGAATCTGGAACAATGGGA GCTTCTGGCACTGGCGGCTTTGGCGGCGGTAACATGGGCG GCATGGGCGGTCAGAATGGT GGCTTCGGCGGTGGCAGACG ASEQ ID NO: 3 SEQ ID NO: 4 GCARNSTSTTTASGGETTIT GGCTGTGCCAGAAATTCAACSAITKEDTDVTHADDAENYR TTCAACTACTACTGCATCAG VSITGDFTVTSDTSDGVTQSGCGGTGAGACTACCATCACT GSVYTITKAGEYTVTGLLSE TCAGCCATTACTAAAGAAGAGQLIVDAGNENEVTIVLNGT CACAGATGTAACACACGCAG SITCSSGSPIYVKNASEVKIATGATGCTGAGAATTACAGA KSEENSFNEVIDNRNEATED GTCTCCATTACAGGTGATTTSSDDAGNAAIYATCDLKLVG CACTGTGACATCTGATACAT KGALVVTGNYNNGIQSKDDLCAGATGGAGTTACACAGTCA SIKNVIVKVTAVNNAVKVND GGTTCTGTATACACAATCACAVDIESGNIIAISAKGDGIK AAAGGCTGGTGAATATACAG TSNSSISNKGNQKGIVTITSTAACAGGACTTTTATCAGAA GNIDIYAACDGIDASYGADI GGACAGCTTATCGTTGACGCSGDGNLNIYTDTYSEYSEEV AGGTAATGAGAACGAGGTTA TSSGSSSGTSSGRDSSANKSCTATCGTATTGAACGGAACA ASANTVSYVATSDTIANAPS TCTATCACATGCTCAAGTGGGFGGGNMGSGNAPDMSNGNA TTCACCTATATACGTTAAAA PNMNGSSDRNKTGGNRPGMPATGCTTCAGAAGTCAAGATT GDFNESGNSSGQSYSTKGIK AAATCAGAAGAGAACTCATTAESEINISGFTINICSTDDG TAACGAAGTAATTGACAATC IHANSDSGVLETGEDGKGTIGTAACGAAGCTACAGAAGAT VINGGSITISSGDDGMHADK TCTTCTGATGACGCTGGCAAQLDVNDGYINVVTSYEGLEA CGCAGCAATCTATGCAACAT MTINLNGGKIYVYATDDGINGCGATTTAAAACTCGTCGGC ACTGDGKTSPIVNVTGGYID AAAGGAGCCTTAGTTGTAACVTTTSGDTDGIDSNGNYVQT AGGTAATTACAATAATGGTA GGFVLVKGGSSSGNVSGSIDTCCAGAGCAAGGATGACCTT VDGTVTITGGTCVALGGVCE TCTATTAAAAATGTGATTGTTPVNSANAYVLGSVSFSSGS TAAAGTTACTGCTGTGAACA YSLKDSSGNEVISFTVDGSFACGCAGTCAAAGTCAACGAT SNGWICSDTLTTGSSYTLYR GCCGTTGATATTGAATCTGGGADSIADWTQE AAATATAATTGCAATCTCCG CTAAAGGCGATGGCATCAAGACTTCTAACAGCAGTATTTC TAACAAGGGCAACCAGAAGG GAATCGTTACAATCACTAGTGGTAACATTGATATTTATGC AGCCTGTGACGGTATAGACG CATCTTACGGGGCAGATATATCAGGTGACGGTAACTTAAA CATTTATACAGATACTTACT CTGAATATAGTGAAGAAGTTACTTCGTCAGGCAGTTCTTC AGGCACGTCTTCTGGCCGGG ACAGCTCTGCTAACAAGTCTGCTTCTGCCAATACTGTTTC TTATGTGGCAACTTCTGACA CTATTGCCAACGCACCTAGCGGCTTTGGTGGTGGCAACAT GGGTAGCGGCAATGCTCCAG ATATGAGTAACGGCAACGCTCCCAATATGAACGGCAGCTC TGATAGAAATAAGACCGGTG GCAACCGTCCAGGAATGCCTGGTGACTTTAATGAATCCGG TAATTCTTCTGGACAGTCCT ACTCAACTAAGGGTATTAAAGCTGAAAGCGAAATAAATAT TTCAGGCTTTACAATTAACA TATGTTCAACAGATGATGGTATCCATGCCAACTCTGACTC AGGTGTACTTGAAACCGGTG AGGACGGCAAAGGAACTATTGTTATCAACGGCGGTTCAAT TACAATTTCTTCTGGCGATG ACGGCATGCACGCTGACAAACAGCTTGATGTCAATGACGG TTACATTAATGTAGTAACTT CATATGAAGGACTTGAGGCTATGACTATCAACTTAAATGG CGGCAAGATATATGTATACG CTACTGATGATGGCATTAATGCCTGCACAGGTGATGGAAA GACTTCTCCAATTGTCAATG TAACTGGTGGATATATAGATGTCACAACTACGTCTGGTGA TACTGATGGTATTGATTCTA ATGGAAATTACGTACAGACCGGTGGATTTGTATTAGTTAA AGGTGGCAGTTCATCTGGAA ATGTATCAGGATCAATTGATGTAGATGGTACCGTAACGAT AACCGGTGGAACATGCGTTG CCCTCGGTGGTGTATGCGAAACACCTGTAAACTCTGCTAA TGCTTATGTATTAGGTTCCG TATCATTCAGTTCTGGAAGCTATTCACTTAAAGATTCTTC TGGCAACGAAGTTATAAGCT TCACTGTTGACGGTTCATTTAGCAACGGCTGGATATGTTC TGACACTCTTACAACCGGCT CAAGCTACACACTCTACCGGGGGGCAGACTCTATTGCAGA CTGGACTCAGGAA SEQ ID NO: 5 SEQ ID NO: 6MGCARNSTSTTTASGGETTI ATGGGCTGTGCCAGAAATTC TSAITKEDTDVTHADDAENYAACTTCAACTACTACTGCAT RVSITGDFTVTSDTSDGVTQ CAGGCGGTGAGACTACCATCSGSVYTITKAGEYTVTGLLS ACTTCAGCCATTACTAAAGA EGQLIVDAGNENEVTIVLNGAGACACAGATGTAACACACG TSITCSSGSPIYVKNASEVK CAGATGATGCTGAGAATTACIKSEENSFNEVIDNRNEATE AGAGTCTCCATTACAGGTGA DSSDDAGNAAIYATCDLKLVTTTCACTGTGACATCTGATA GKGALVVTGNYNNGIQSKDD CATCAGATGGAGTTACACAGLSIKNVIVKVTAVNNAVKVN TCAGGTTCTGTATACACAAT DAVDIESGNIIAISAKGDGICACAAAGGCTGGTGAATATA KTSNSSISNKGNQKGIVTIT CAGTAACAGGACTTTTATCASGNIDIYAACDGIDASYGAD GAAGGACAGCTTATCGTTGA ISGDGNLNIYTDTYSEYSEECGCAGGTAATGAGAACGAGG VTSSGSSSGTSSGRDSSANK TTACTATCGTATTGAACGGASASANTVSYVATSDTIANAP ACATCTATCACATGCTCAAG SGFGGGNMGSGNAPDMSNGNTGGTTCACCTATATACGTTA APNMNGSSDRNKTGGNRPGM AAAATGCTTCAGAAGTCAAGPGDFNESGNSSGQSYSTKGI ATTAAATCAGAAGAGAACTC KAESEINISGFTINICSTDDATTTAACGAAGTAATTGACA GIHANSDSGVLETGEDGKGT ATCGTAACGAAGCTACAGAAIVINGGSITISSGDDGMHAD GATTCTTCTGATGACGCTGG KQLDVNDGYINVVTSYEGLECAACGCAGCAATCTATGCAA AMTINLNGGKIYVYATDDGI CATGCGATTTAAAACTCGTCNACTGDGKTSPIVNVTGGYI GGCAAAGGAGCCTTAGTTGT DVTTTSGDTDGIDSNGNYVQAACAGGTAATTACAATAATG TGGFVLVKGGSSSGNVSGSI GTATCCAGAGCAAGGATGACDVDGTVTITGGTCVALGGVC CTTTCTATTAAAAATGTGAT ETPVNSANAYVLGSVSFSSGTGTTAAAGTTACTGCTGTGA SYSLKDSSGNEVISFTVDGS ACAACGCAGTCAAAGTCAACFSNGWICSDTLTTGSSYTLY GATGCCGTTGATATTGAATC RGADSIADWTQETGGAAATATAATTGCAATCT CCGCTAAAGGCGATGGCATC AAGACTTCTAACAGCAGTATTTCTAACAAGGGCAACCAGA AGGGAATCGTTACAATCACT AGTGGTAACATTGATATTTATGCAGCCTGTGACGGTATAG ACGCATCTTACGGGGCAGAT ATATCAGGTGACGGTAACTTAAACATTTATACAGATACTT ACTCTGAATATAGTGAAGAA GTTACTTCGTCAGGCAGTTCTTCAGGCACGTCTTCTGGCC GGGACAGCTCTGCTAACAAG TCTGCTTCTGCCAATACTGTTTCTTATGTGGCAACTTCTG ACACTATTGCCAACGCACCT AGCGGCTTTGGTGGTGGCAACATGGGTAGCGGCAATGCTC CAGATATGAGTAACGGCAAC GCTCCCAATATGAACGGCAGCTCTGATAGAAATAAGACCG GTGGCAACCGTCCAGGAATG CCTGGTGACTTTAATGAATCCGGTAATTCTTCTGGACAGT CCTACTCAACTAAGGGTATT AAAGCTGAAAGCGAAATAAATATTTCAGGCTTTACAATTA ACATATGTTCAACAGATGAT GGTATCCATGCCAACTCTGACTCAGGTGTACTTGAAACCG GTGAGGACGGCAAAGGAACT ATTGTTATCAACGGCGGTTCAATTACAATTTCTTCTGGCG ATGACGGCATGCACGCTGAC AAACAGCTTGATGTCAATGACGGTTACATTAATGTAGTAA CTTCATATGAAGGACTTGAG GCTATGACTATCAACTTAAATGGCGGCAAGATATATGTAT ACGCTACTGATGATGGCATT AATGCCTGCACAGGTGATGGAAAGACTTCTCCAATTGTCA ATGTAACTGGTGGATATATA GATGTCACAACTACGTCTGGTGATACTGATGGTATTGATT CTAATGGAAATTACGTACAG ACCGGTGGATTTGTATTAGTTAAAGGTGGCAGTTCATCTG GAAATGTATCAGGATCAATT GATGTAGATGGTACCGTAACGATAACCGGTGGAACATGCG TTGCCCTCGGTGGTGTATGC GAAACACCTGTAAACTCTGCTAATGCTTATGTATTAGGTT CCGTATCATTCAGTTCTGGA AGCTATTCACTTAAAGATTCTTCTGGCAACGAAGTTATAA GCTTCACTGTTGACGGTTCA TTTAGCAACGGCTGGATATGTTCTGACACTCTTACAACCG GCTCAAGCTACACACTCTAC CGGGGGGCAGACTCTATTGCAGACTGGACTCAGGAA SEQ ID NO: 7 SEQ ID NO: 8 GCARNSTSTTTASGGETTITGGCTGTGCCAGAAATTCAAC SAITKEDTDVTHADDAENYR TTCAACTACTACTGCATCAGVSITGDFTVTSDTSDGVTQS GCGGTGAGACTACCATCACT GSVYTITKAGEYTVTGLLSETCAGCCATTACTAAAGAAGA GQLIVDAGNENEVTIVLNGT CACAGATGTAACACACGCAGSITCSSGSPIYVKNASEVKI ATGATGCTGAGAATTACAGA KSEENSFNEVIDNRNEATEDGTCTCCATTACAGGTGATTT SSDDAGNAAIYATCDLKLVG CACTGTGACATCTGATACATKGALVVTGNYNNGIQSKDDL CAGATGGAGTTACACAGTCA SIKNVIVKVTAVNNAVKVNDGGTTCTGTATACACAATCAC AVDIESGNIIAISAKGDGIK AAAGGCTGGTGAATATACAGTSNSSISNKGNQKGIVTITS TAACAGGACTTTTATCAGAA GNIDIYAACDGIDASYGADIGGACAGCTTATCGTTGACGC SGDGNLNIYTDTYSEYSEEV AGGTAATGAGAACGAGGTTATSSGSSSGTSSGRDSSANKS CTATCGTATTGAACGGAACA ASANTVSYVATSDTIANAPSTCTATCACATGCTCAAGTGG GFGGGNMGSGNAPDMSNGNA TTCACCTATATACGTTAAAAPNMNGSSDRNKTGGNRPGMP ATGCTTCAGAAGTCAAGATT GDFNESGNSSGQSYSTKGIKAAATCAGAAGAGAACTCATT AESEINISGFTINICSTDDG TAACGAAGTAATTGACAATCIHANSDSGVLETGEDGKGTI GTAACGAAGCTACAGAAGAT VINGGSITISSGDDGMHADKTCTTCTGATGACGCTGGCAA QLDVNDGYINVVTSYEGLEA CGCAGCAATCTATGCAACATMTINLNGGKIYVYATDDGIN GCGATTTAAAACTCGTCGGC ACTGDGKTSPIVNVTGGYIDAAAGGAGCCTTAGTTGTAAC VTTTSGDTDGIDSNGNYVQT AGGTAATTACAATAATGGTAGGFVLVKGGSSSGNVSGSID TCCAGAGCAAGGATGACCTT VDGTVTITGGTCVALGGVCETCTATTAAAAATGTGATTGT TPVNSANAYVLGSVSFSSGS TAAAGTTACTGCTGTGAACAYSLKDSSGNEVISFTVDGSF ACGCAGTCAAAGTCAACGAT SNGWICSDTLTTGSSYTLYRGCCGTTGATATTGAATCTGG GADSIADWTQESGTMGASGT AAATATAATTGCAATCTCCGGGFGGGNMGGMGGQNGGFGG CTAAAGGCGATGGCATCAAG GRR ACTTCTAACAGCAGTATTTCTAACAAGGGCAACCAGAAGG GAATCGTTACAATCACTAGT GGTAACATTGATATTTATGCAGCCTGTGACGGTATAGACG CATCTTACGGGGCAGATATA TCAGGTGACGGTAACTTAAACATTTATACAGATACTTACT CTGAATATAGTGAAGAAGTT ACTTCGTCAGGCAGTTCTTCAGGCACGTCTTCTGGCCGGG ACAGCTCTGCTAACAAGTCT GCTTCTGCCAATACTGTTTCTTATGTGGCAACTTCTGACA CTATTGCCAACGCACCTAGC GGCTTTGGTGGTGGCAACATGGGTAGCGGCAATGCTCCAG ATATGAGTAACGGCAACGCT CCCAATATGAACGGCAGCTCTGATAGAAATAAGACCGGTG GCAACCGTCCAGGAATGCCT GGTGACTTTAATGAATCCGGTAATTCTTCTGGACAGTCCT ACTCAACTAAGGGTATTAAA GCTGAAAGCGAAATAAATATTTCAGGCTTTACAATTAACA TATGTTCAACAGATGATGGT ATCCATGCCAACTCTGACTCAGGTGTACTTGAAACCGGTG AGGACGGCAAAGGAACTATT GTTATCAACGGCGGTTCAATTACAATTTCTTCTGGCGATG ACGGCATGCACGCTGACAAA CAGCTTGATGTCAATGACGGTTACATTAATGTAGTAACTT CATATGAAGGACTTGAGGCT ATGACTATCAACTTAAATGGCGGCAAGATATATGTATACG CTACTGATGATGGCATTAAT GCCTGCACAGGTGATGGAAAGACTTCTCCAATTGTCAATG TAACTGGTGGATATATAGAT GTCACAACTACGTCTGGTGATACTGATGGTATTGATTCTA ATGGAAATTACGTACAGACC GGTGGATTTGTATTAGTTAAAGGTGGCAGTTCATCTGGAA ATGTATCAGGATCAATTGAT GTAGATGGTACCGTAACGATAACCGGTGGAACATGCGTTG CCCTCGGTGGTGTATGCGAA ACACCTGTAAACTCTGCTAATGCTTATGTATTAGGTTCCG TATCATTCAGTTCTGGAAGC TATTCACTTAAAGATTCTTCTGGCAACGAAGTTATAAGCT TCACTGTTGACGGTTCATTT AGCAACGGCTGGATATGTTCTGACACTCTTACAACCGGCT CAAGCTACACACTCTACCGG GGGGCAGACTCTATTGCAGACTGGACTCAGGAATCTGGAA CAATGGGAGCTTCTGGCACT GGCGGCTTTGGCGGCGGTAACATGGGCGGCATGGGCGGTC AGAATGGTGGCTTCGGCGGT GGCAGACGA

Epithelial Barrier Function in Disease

Studies in recent years have identified a major role of both genetic andenvironmental factors in the pathogenesis of IBD. Markus Neurath,“Cytokines in Inflammatory Bowel Disease,” Nature Reviews Immunology,Vol. 14., 329-342(2014). A combination of these IBD risk factors seemsto initiate detrimental changes in epithelial barrier function, therebyallowing the translocation of luminal antigens (for example, bacterialantigens from the commensal microbiota) into the bowel wall. Id.Subsequently, aberrant and excessive responses, such as increasedpro-inflammatory cytokine release, to such environmental triggers causesubclinical or acute mucosal inflammation in a genetically susceptiblehost. Id. Thus, the importance of proper epithelial barrier function inIBD is apparent, for in subjects that fail to resolve acute intestinalinflammation, chronic intestinal inflammation develops that is inducedby the uncontrolled activation of the mucosal immune system. Inparticular, mucosal immune cells, such as macrophages, T cells, and thesubsets of innate lymphoid cells (ILCs), seem to respond to microbialproducts or antigens from the commensal microbiota by, e.g., producingcytokines that can promote chronic inflammation of the gastrointestinaltract. Consequently, restoring proper epithelial barrier function topatients may be critical in resolving IBD.

The therapeutic activity of SG-14 was identified in part by itsbeneficial effects on epithelial barrier function both in vitro and invivo. As shown in Example 2, SG-14 (comprising SEQ ID NO:3) increasedepithelial barrier integrity as shown by an in vitro trans-epithelialelectrical resistance (TEER) assay. TEER assays are well-known methodsfor measuring effects on the structural and functional integrity of anepithelial cell layer (Srinivasan et al., 2015, J Lab Autom, 20:107-126;Beduneau et al., 2014, Eur J Pharm Biopharm, 87:290-298; Zolotarevsky etal., 2002, Gastroenterology, 123:163-172; Dewi, et al. (2004) J. Virol.Methods. 121:171-180, and in Mandic, et al. (2004) Clin. Exp. Metast.21:699-704.). The assay performed and described herein consists of anepithelial monolayer made up of enterocyte and goblets cells to moreaccurately model the structural and functional components of theintestinal epithelium. The cells are cultured until tight junctionformation occurs and barrier function capacity is assessed by ameasurement of trans-epithelial electrical resistance. Upon addition ofan insult, such as heat killed E. coli, there is a decrease inelectrical resistance across the epithelial layer. Control reagentsuseful in the TEER assay include staurosporine and a myosin light chainkinase inhibitor. Staurosporine is a broad spectrum kinase inhibitor,originating from Streptomyces staurosporeus, which induces apoptosis.This reagent disrupts about 98% of the gap junctions leading to adecrease in electrical resistance in a TEER assay. Myosin light chainkinase (MLCK) is the terminal effector in a signaling cascade induced bypro-inflammatory cytokines, which results in contraction of theperijunctional actomyosin ring, resulting in separation of the gapjunctions. By inhibiting MLCK, disruption of tight junctions isprevented. MLCK inhibitor in a TEER assay should reduce or prevent thereduction of electrical resistance in a TEER assay.

In some embodiments, the SG-14 protein or variant or fragment thereof asdescribed herein can be characterized by its ability to increaseepithelial barrier function integrity as assessed by an in vitro TEERassay. The SG-14 protein or variant or fragment thereof may increaseelectrical resistance in a TEER assay by at least about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or 90% as compared to the TEER assayperformed in the absence of the protein.

In addition, Example 6 shows that SG-14 protein can enhance orfacilitate epithelial wound healing, an activity that can play a role inthe maintenance or repair of and epithelial barrier such as anintestinal or mucosal epithelial barrier.

In view of the effect of SG-14 to repair barrier function integrity invitro, SG-14 was analyzed in vivo for its ability to reduce damage in arodent model of IBD. Example 7 (SG-14) describes studies done using aDSS (dextran sodium sulfate) animal model, a model well accepted for thestudy of agents on IBDs (Chassaign et al., 2014, Curr Protoc Imunol,104:Unit-15.25; Kiesler et al., 2015, Cell Mol Gastroenterol Hepatol).DSS is a sulfated polysaccharide that is directly toxic to colonicepithelium and causes epithelial cell injury leading to loss of barrierfunction due to disrupted gap junctions. In these experiments, animalswere treated with SG-14 prior to induction of colitis in the mouse. As apositive control, the mice were also treated with Gly2-GLP2, a stableanalog of glucagon-like peptide 2 (GLP2). Gly2-GLP2 is known to promoteepithelial cell growth and reduce colonic injury in experiment mousecolitis models. Results of the DSS studies show that SG-14 protein waseffective in reducing epithelial barrier permeability (see, e.g., FIG. 6). SG-14 treatment also reduced scores in gross pathology (see, e.g.,FIG. 9 ).

Variants of SG-14

In another example, certain amino acids of the taught proteins may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, binding sites on substrate molecules, receptors,antigen-binding regions of antibodies, and the like. Thus, theseproteins would be biologically functional equivalents of the disclosedproteins (e.g. comprising SEQ ID NO:3 or variants thereof). In someembodiments, “conservative” changes do not disrupt the biologicalactivity of the protein, as the structural change is not one thatimpinges on the protein's ability to carry out its designed function. Itis thus contemplated by the inventors that various changes may be madein the sequence of genes and proteins disclosed herein, while stillfulfilling the goals of the present disclosure.

In some embodiments, a modified or variant protein is provided whichcontains at least one non-naturally occurring amino acid substitutionrelative to SEQ ID NO:3. In other embodiments, the variant proteincomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutionsrelative to SEQ ID NO:3 or SEQ ID NO:5.

In some embodiments, the taught proteins have markedly differentstructural and/or functional characteristics, as compared to a proteincomprising or consisting of SEQ ID NO:3.

The term “SG-14 variant” as used herein can include SG-14 proteins thatare, e.g., identical to not identical to a protein comprising thesequence of SEQ ID NO:3 and which are further modified such as by a PTMor fusion or linkage to a second agent, e.g., a protein or peptide.

Protein PTMs occur in vivo and can increase the functional diversity ofthe proteome by the covalent addition of functional groups or proteins,proteolytic cleavage of regulatory subunits or degradation of entireproteins. Isolated proteins prepared according to the present disclosurecan undergo 1 or more PTMs in vivo or in vitro. The type ofmodification(s) depends on host cell in which the protein is expressedand includes but is not limited to phosphorylation, glycosylation,ubiquitination, nitrosylation (e.g., S-nitrosylation), methylation,acetylation (e.g., N-acetylation), lipidation (myristoylation,N-myristoylation, S-palmitoylation, famesylation, S-prenylation,S-palmitoylation) and proteolysis may influence almost all aspects ofnormal cell biology and pathogenesis. The isolated and/or purified SG-14proteins or variants or fragments thereof as disclosed herein maycomprise one or more the above recited post-translational modifications.

The SG-14 protein or variant or fragment thereof may be a fusion proteinin which the N- and/or C-terminal domain is fused to a second proteinvia a peptide bond. Commonly used fusion partners well known to theordinarily skilled artisan include but are not limited to human serumalbumin and the crystallizable fragment, or constant domain of IgG, Fc.In some embodiments, the SG-14 protein or variant or fragment thereof islinked to a second protein or peptide via a disulfide bond, wherein thesecond protein or peptide comprises a cysteine residue.

As aforementioned, modifications and/or changes (e.g., substitutions,insertions, deletions) may be made in the structure of proteinsdisclosed herein. Thus, the present disclosure contemplates variation insequence of these proteins, and nucleic acids coding therefore, wherethey are nonetheless able to retain substantial activity with respect tothe functional activities assessed in various in vitro and in vivoassays as well as in therapeutic aspects of the present disclosure. Interms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity.

It is also contemplated that the SG-14 protein or variant or fragmentthereof is one which, when administered to a subject, can reducedisease-associated weight loss, improve the clinical pathology score,and/or minimize colon shortening in the subject. In some embodiments,the subject is a mammal which has genetically or clinically inducedinflammatory disorder or dysfunctional epithelial barrier function.Alternatively, the animal has an idiopathic gastrointestinal disorderinvolving a decrease in epithelial barrier function or intestinalinflammatory disorder. In other embodiments, the mammal is a human,non-human primate, or a rodent. The rodent may be a mouse or rat.

In some embodiments, the SG-14 protein or variant or fragment thereofaccording to the present disclosure is one which can modulate productionof and/or secretion of a cytokine in an in vitro assay or in an subjectadministered the protein. In some embodiments, secretion of cytokines isreduced in vitro. Levels of cytokines produced and/or secreted in an invitro assay or subject administered the protein are likely to bemeasured in the blood, serum, and/or plasma of the subject.Administration of the protein may result in a decrease in the serumlevels of a pro-inflammatory cytokine such as one or more of TNF-α,IL-17, IL-1β, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1,MIP-3α, CXCL1, and IL-23. Alternatively, the cytokine may be ananti-inflammatory cytokine, in which case administration of the proteinresults in an increase in serum levels of an anti-inflammatory cytokinesuch as IL-4, IL-10, IL-13, IFN-α, and TGF-β.

In some embodiments, the SG-14 protein or variant or fragment thereofhas the functional ability to reduce gastrointestinal inflammation whenadministered to a subject such as a mammal (e.g., rodent, non humanprimate, or human). In other embodiments, the protein has the functionalability to reduce inflammatory (i.e. pro-inflammatory) cytokines, whenadministered to the subject. In yet other embodiments, the protein isable to reduce TNF-α and/or IL-23, when administered to the subject. Instill other embodiments, the protein has the functional ability toincrease anti-inflammatory cytokines, when administered to the subject.In some aspects, a protein of the disclosure is able to increase IL-10,when administered to the subject.

A SG-14 protein or variant or fragment thereof according to the presentdisclosure is one which, when administered to a subject (e.g., rodent,non human primate, or human), can improve gastrointestinal epithelialcell barrier function, reduce disease-associated weight loss, improveclinical scores, improve colon length and/or colon weight-to-lengthreadouts, induce or increase mucin gene expression (e.g., muc2expression), increase the structural integrity and/or functionality of agastrointestinal mucous barrier (e.g., in the small intestine, largeintestine, mouth and/or esophagus), and/or reduce inflammation in thegastrointestinal tract.

In some embodiments, the SG-14 protein or variant or fragment thereofresulting from such a substitution, insertion and/or deletion of aminoacids relative to SEQ ID NO: 3 or SEQ ID NO: 5 maintains a level offunctional activity which is substantially the same as that of a proteinof SEQ ID NO: 3 (e.g., is able to decrease epithelial barrierpermeability in a DSS mouse model). The variant protein may be useful asa therapeutic for treatment or prevention of a variety of conditions,including, but not limited to inflammatory conditions and/or barrierfunction disorders, including, but not limited to, inflammation of thegastrointestinal (including oral, esophageal, and intestinal) mucosa,impaired intestinal epithelial cell gap junction integrity. In someembodiments, the modified protein has one or more of the followingeffects when administered to an individual suffering from, orpredisposed to, an inflammatory condition and/or barrier functiondisorder: improvement of epithelial barrier integrity, e.g., followinginflammation induced disruption; suppression of production of at leastone pro-inflammatory cytokine (e.g., TNF-α and/or IL-23) by one or moreimmune cell(s); induction of mucin production in epithelial cells;and/or improvement of epithelial wound healing. Moreover, the modifiedor variant SG-14 protein may be used for treatment or prevention of adisorder or condition such as, but not limited to, inflammatory boweldisease, ulcerative colitis, Crohn's disease, short bowel syndrome, GImucositis, oral mucositis, chemotherapy-induced mucositis,radiation-induced mucositis, necrotizing enterocolitis, pouchitis, ametabolic disease, celiac disease, inflammatory bowel syndrome, orchemotherapy associated steatohepatitis (CASH).

As demonstrated, e.g., in Example 6, the SG-14 protein can enhanceepithelial wound healing. Accordingly, provided herein is a therapeuticprotein comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:5 or a variant or fragment thereof, wherein the protein can increasewound healing in an in vitro assay.

Methods of Treatment

The SG-14 proteins described herein including variants (e.g., amino acidsubstitutions, deletions, insertions), modifications (e.g.,glycosylation, acetylation), SG-14 fragments and fusions thereof arecontemplated for use in treating a subject diagnosed with or sufferingfrom a disorder related to inflammation within the gastrointestinaltract and/or malfunction of epithelial barrier function within thegastrointestinal tract.

Provided herein are methods for treating a subject in need thereofcomprising administering to the subject a pharmaceutical compositioncomprising a SG-14 protein or fragment or variant thereof as describedin the present disclosure. The subject can be one who has been diagnosedwith inflammatory bowel disease, ulcerative colitis, pediatric UC,Crohn's disease, pediatric Crohn's disease, short bowel syndrome,mucositis GI mucositis, oral mucositis, mucositis of the esophagus,stomach, small intestine (duodenum, jejunum, ileum), large intestine(colon), and/or rectum, chemotherapy-induced mucositis,radiation-induced mucositis, necrotizing enterocolitis, pouchitis, ametabolic disease, celiac disease, irritable bowel syndrome, orchemotherapy associated steatohepatitis (CASH) Administration of theSG-14 pharmaceutical compositions described herein may also be usefulfor wound healing applications.

Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) classically includes ulcerative colitis(UC) and Crohn's disease (CD). The pathogenesis of inflammatory boweldisease is not known. A genetic predisposition has been suggested, and ahost of environmental factors, including bacterial, viral and, perhaps,dietary antigens, can trigger an ongoing enteric inflammatory cascade.Id. IBD can cause severe diarrhea, pain, fatigue, and weight loss. IBDcan be debilitating and sometimes leads to life-threateningcomplications. Accordingly, in some embodiments, the method of treatmentas described herein is effective to reduce, prevent or eliminate any oneor more of the symptoms described above wherein the method comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising the SG-14 protein orvariant or fragment thereof. In some embodiments, the method oftreatment results in remission.

Ulcerative Colitis

Ulcerative colitis is an inflammatory bowel disease that causeslong-lasting inflammation and sores (ulcers), in the innermost lining ofyour large intestine (colon) and rectum. Ulcerative colitis typicallypresents with shallow, continuous inflammation extending from the rectumproximally to include, in many patients, the entire colon. Fistulas,fissures, abscesses and small-bowel involvement are absent. Patientswith limited disease (e.g., proctitis) typically have mild butfrequently recurrent symptoms, while patients with pancolitis morecommonly have severe symptoms, often requiring hospitalization. Botomanet al., “Management of Inflammatory Bowel Disease,” Am. Fam. Physician,Vol. 57(1):57-68 (Jan. 1, 1998) (internal citations omitted). Thus,ulcerative colitis is an IBD that causes long-lasting inflammation andsores (ulcers) in the innermost lining of your large intestine (colon)and rectum.

Crohn's Disease

Unlike ulcerative colitis, Crohn's disease can involve the entireintestinal tract, from the mouth to the anus, with discontinuous focalulceration, fistula formation and perianal involvement. The terminalileum is most commonly affected, usually with variable degrees ofcolonic involvement. Subsets of patients have perianal disease withfissures and fistula formation. Only 2 to 3 percent of patients withCrohn's disease have clinically significant involvement of the uppergastrointestinal tract. Botoman et al., “Management of InflammatoryBowel Disease,” Am. Fam. Physician, Vol. 57(1):57-68 (Jan. 1, 1998)(internal citations omitted). Thus, Crohn's disease is an IBD thatcauses inflammation of the lining of your digestive tract. In Crohn'sdisease, inflammation often spreads deep into affected tissues. Theinflammation can involve different areas of the digestive tract, i.e.the large intestine, small intestine, or both. Collagenous colitis andlymphocytic colitis also are considered inflammatory bowel diseases, butare usually regarded separately from classic inflammatory bowel disease.

Clinical Parameters of Inflammatory Bowel Disease

As previously discussed, inflammatory bowel disease encompassesulcerative colitis and Crohn's disease. There are numerous scores andclinical markers known to one of skill in the art that can be utilizedto access the efficacy of the administered proteins described herein intreating these conditions.

There are two general approaches to evaluating patients with IBD. Thefirst involves the visual examination of the mucosa and relies on theobservation of signs of damage to the mucosa, in view of the fact thatIBD is manifested by the appearance of inflammation and ulcers in the GItract. Any procedure that allows an assessment of the mucosa can beused. Examples include barium enemas, x-rays, and endoscopy. Anendoscopy may be of the esophagus, stomach and duodenum(esophagogastroduodenoscopy), small intestine (enteroscopy), or largeintestine/colon (colonoscopy, sigmoidoscopy). These techniques are usedto identify areas of inflammation, ulcers and abnormal growths such aspolyps.

Scoring systems based on this visual examination of the GI tract existto determine the status and severity of IBD, and these scoring systemsare intended to ensure that uniform assessment of different patientsoccurs, despite the fact that patients may be assessed by differentmedical professionals, in diagnosis and monitoring of these diseases aswell as in clinical research evaluations. Examples of evaluations basedon visual examination of UC are discussed and compared in Dapemo M et al(J Crohns Colitis. 2011 5:484-98).

Clinical scoring systems also exist, with the same purpose. The findingson endoscopy or other examination of the mucosa can be incorporated intothese clinical scoring systems, but these scoring systems alsoincorporate data based on symptoms such as stool frequency, rectalbleeding and physician's global assessment. IBD has a variety ofsymptoms that affect quality of life, so certain of these scoringsystems also take into account a quantitative assessment of the effecton quality of life as well as the quantification of symptoms.

One example of a scoring system for UC is the Mayo scoring system(Schroeder et al., N Eng J Med, 1987, 317:1625-1629), but others existthat have less commonly been used and include the Ulcerative ColitisEndoscopic Index of Severity (UCEIS) score (Travis et al, 2012, Gut,61:535-542), Baron Score (Baron et al., 1964, BMJ, 1:89), UlcerativeColitis Colonoscopic Index of Severity (UCCIS) (Thia et al., 2011,Inflamm Bowel Dis, 17:1757-1764), Rachmilewitz Endoscopic Index(Rachmilewitz, 1989, BMJ, 298:82-86), Sutherland Index (also known asthe UC Disease Activity Index (UCDAI) scoring system; Sutherland et al.,1987, Gastroenterology, 92:1994-1998), Matts Score (Matts, 1961, QJM,30:393-407), and Blackstone Index (Blackstone, 1984, Inflammatory boweldisease. In: Blackstone M O (ed.) Endoscopic interpretation: normal andpathologic appearances of the gastrointestinal tract, 1984, pp.464-494). For a review, see Paine, 2014, Gastroenterol Rep 2:161-168.Accordingly, also contemplated herein is a method for treating a subjectdiagnosed with and suffering from UC, wherein the treatment comprisesadministering a SG-14 protein or variant or fragment thereof asdescribed herein and wherein the treatment results in a decrease in theUC pathology as determined by measurement of the UCEIS score, the Baronscore, the UCCIS score, the Rachmilewitz Endoscopic Index, theSutherland Index, and/or the Blackstone Index.

An example of a scoring system for CD is the Crohn's Disease ActivityIndex (CDAI) (Sands B et al 2004, N Engl J Med 350 (9): 876-85); mostmajor studies use the CDAI in order to define response or remission ofdisease. Calculation of the CDAI score includes scoring of the number ofliquid stools over 7 days, instances and severity of abdominal pain over7 days, general well-being over 7 days, extraintestinal complications(e.g., arthritis/arthralgia, iritis/uveitis, erythema nodosum, pyodermagangrenosum, aphtous stomatitis, anal fissure/fistula/abscess, and/orfever >37.8° C.), use of antidiarrheal drugs over 7 days, present ofabdominal mass, hematocrit, and body weight as a ratio of ideal/observedor percentage deviation from standard weight. Based on the CDAI score,the CD is classified as either asymptomatic remission (0 to 149 points),mildly to moderately active CD (150 to 220 points), moderately toseverely active CD (221 to 450 points), or severely active fulminantdisease (451 to 1000 points). In some embodiments, the method oftreatment comprising administering to a patient diagnosed with CD atherapeutically effective amount of SG-14 protein or variant or fragmentthereof results in a decrease in a diagnostic score of CD. For example,the score may change the diagnosis from severely active to mildly ormoderately active or to asymptomatic remission.

The Harvey-Bradshaw index is a simpler version of the CDAI whichconsists of only clinical parameters (Harvey et al., 1980, Lancet1(8178):1134-1135). The impact on quality of life is also addressed bythe Inflammatory Bowel Disease Questionnaire (IBDQ) (Irvine et al.,1994, Gastroenterology 106: 287-296). Alternative methods furtherinclude CDEIS and SES CD (see, e.g., Levesque, et al. (2015)Gastroentrol. 148:37 57).

In some embodiments, a method of treating an IBD, e.g., UC, is providedwherein the treatment is effective in reducing the Mayo Score. The MayoScore is a combined endoscopic and clinical scale used to assess theseverity of UC and has a scale of 1-12 The Mayo Score is a composite ofsubscores for stool frequency, rectal bleeding, findings of flexibleproctosigmoidoscopy or colonoscopy, and physician's global assessment(Paine, 2014, Gastroenterol Rep 2:161-168). With respect to rectalbleeding, blood streaks seen in the stool less than half the time isassigned 1 point, blood in most stools is assigned 2 points and pureblood passed is assigned 3 points. Regarding stool frequency, a normalnumber of daily stools is assigned 0 points, 1 or 2 more stools thannormal is assigned 1 point, 3 or 4 more stools than normal is assigned 2points, and 5 or more stools than usual is assigned 3 points. Withrespect to the endoscopy component, a score of 0 indicates normal mucosaor inactive UC, a score of 1 is given for mild disease with evidence ofmild friability, reduced vascular pattern, and mucosal erythema, a scoreof 2 is given for moderate disease with friability, erosions, completeloss of vascular pattern, and significant erythema, and a score of 3 isgiven for ulceration and spontaneous bleeding (Schroeder et al., 1987, NEngl J Med, 317:1625-1629). Global assessment by a physician assigns 0points for a finding of normal, 1 point for mild colitis, 2 points formoderate colitis and 3 points for severe colitis. Accordingly, in someembodiments, a patient treated with a SG-14 therapeutic protein orvariant or fragment thereof is successfully treated when the patientexperiences a reduction in the Mayo Score by at least 1, 2 or 3 pointsin at least one of: rectal bleeding, blood streaks seen in the stool,endoscopy subscore and physician's global assessment. In someembodiments, the method of treatment comprising administering to apatient diagnosed with UC a therapeutically effective amount of SG-14protein or variant or fragment thereof results in a decrease in adiagnostic score of UC. For example, the score may change a diagnosticscore, e.g., Mayo Score, by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11points.

Pouchitis

Additionally or alternatively, the compositions comprising a SG-14therapeutic protein or variant and methods of administration asdescribed herein can be used to treat pouchitis. Pouchitis is aninflammation of the lining of a pouch that is surgically created in thetreatment of UC. Specifically, subjects having serious UC may have theirdiseased colon removed and the bowel reconnected by a procedure calledileoanal anastomosis (IPAA) or J-pouch surgery. Pouchitis cases canrecur in many patients, manifesting either as acute relapsing pouchitisor chronic, unremitting pouchitis. Accordingly, provided herein aremethods for treating pouchitis, acute pouchitis or recurrent pouchitis.

Pouchitis activity can be classified as remission (no active pouchitis),mild to moderately active (increased stool frequency, urgency, and/orinfrequent incontinence), or severely active (frequent incontinenceand/or the patient is hospitalized for dehydration). The duration ofpouchitis can be defined as acute (less than or equal to four weeks) orchronic (four weeks or more) and the pattern classified as infrequent(1-2 acute episodes), relapsing (three or fewer episodes) or continuous.The response to medical treatment can be labeled as treatment responsiveor treatment refractory, with the medication for either case beingspecified. Accordingly, in some embodiments, a method for treating asubject diagnosed with pouchitis is provided wherein treatment with apharmaceutical composition comprising SG-14 or variant or fragmentthereof results in a decrease in the severity of the pouchitis and/orresults in remission.

Mucositis and Mucosal Barriers

The mucosa of the gastrointestinal (GI) tract is a complexmicroenvironment involving an epithelial barrier, immune cells, andmicrobes. A delicate balance is maintained in the healthy colon. Luminalmicrobes are physically separated from the host immune system by abarrier consisting of epithelium and mucus. The pathogenesis of IBD,although not fully elucidated, may involve an inappropriate hostresponse to an altered commensal flora with a dysfunctional mucousbarrier. See, Boltin et al., “Mucin Function in Inflammatory BowelDisease an Update,” J. Clin. Gastroenterol., Vol. 47(2):106-111(February 2013).

Mucositis occurs when cancer treatments (particularly chemotherapy andradiation) break down the rapidly divided epithelial cells lining theintestinal tract (which goes from the mouth to the anus), leaving themucosal tissue open to ulceration and infection. Mucosal tissue, alsoknown as mucosa or the mucous membrane, lines all body passages thatcommunicate with the air, such as the respiratory and alimentary tracts,and have cells and associated glands that secrete mucus. The part ofthis lining that covers the mouth, called the oral mucosa, is one of themost sensitive parts of the body and is particularly vulnerable tochemotherapy and radiation. The oral cavity is the most common locationfor mucositis. While the oral mucosa is the most frequent site ofmucosal toxicity and resultant mucositis, it is understood thatmucositis can also occur along the entire alimentary tract including theesophagus, stomach, small intestine (duodenum, jejunum, ileum), largeintestine (colon), and rectum. In some embodiments, a pharmaceuticalcomposition comprising SG-14 or a variant or fragment thereof istherapeutically effective to treat mucositis of the mouth, esophagus,stomach, small intestine (duodenum, jejunum, ileum), large intestine(colon), and/or rectum.

Oral mucositis can lead to several problems, including pain, nutritionalproblems as a result of inability to eat, and increased risk ofinfection due to open sores in the mucosa. It has a significant effecton the patient's quality of life and can be dose-limiting (i.e.,requiring a reduction in subsequent chemotherapy doses). The WorldHealth Organization has an oral toxicity scale for diagnosis of oralmucositis: Grade 1: soreness erythema, Grade 2: erythema, ulcers;patient can swallow solid food; Grade 3: ulcers with extensive erythema;patient cannot swallow solid food; Grade 4: mucositis to the extent thatalimentation is not possible. Grade 3 and Grade 4 oral mucositis isconsidered severe mucositis. Accordingly, provided herein is a methodfor treating a subject diagnosed with oral mucositis, whereinadministration of a pharmaceutical composition comprising SG-14 or avariant or fragment thereof reduces the grade of oral toxicity by atleast 1 point of the grade scale of 1 to 4.

Colon Shortening

Ulcerative colitis is an idiopathic inflammatory bowel disease thataffects the colonic mucosa and is clinically characterized by diarrhea,abdominal pain and hematochezia. The extent of disease is variable andmay involve only the rectum (ulcerative proctitis), the left side of thecolon to the splenic flexure, or the entire colon (pancolitis). Theseverity of the disease may also be quite variable histologically,ranging from minimal to florid ulceration and dysplasia. Carcinoma maydevelop. The typical histological (microscopic) lesion of ulcerativecolitis is the crypt abscess, in which the epithelium of the cryptbreaks down and the lumen fills with polymorphonuclear cells. The laminapropria is infiltrated with leukocytes. As the crypts are destroyed,normal mucosal architecture is lost and resultant scarring shortens andcan narrow the colon. Thus, colon shortening can be a consequence ofcolitis disease and is often used diagnostically. For example,non-invasive plain abdominal x-rays can demonstrate the gaseous outlineof the transverse colon in the acutely ill patient. Shortening of thecolon and loss of haustral markings can also be demonstrated by plainfilms, as well as a double-contrast barium enema. Indications ofulcerative disease include loss of mucosal detail, cobblestone fillingdefects, and segmental areas of involvement. See, “Ulcerative Colitis:Introduction—Johns Hopkins Medicine,” found at:www.hopkinsmedicine.org/gastroenterology_hepatology/_pdfs/small_large_intestine/ulcerative_colitis.pdf.

Further, art recognized in vivo models of colitis will utilizeshortening of colon length in scoring the severity of colitis in themodel. See, Kim et al., “Investigating Intestinal Inflammation inDSS-induced Model of IBD,” Journal of Visualized Experiments, Vol. 60,pages 2-6 (February 2012).

Epithelial Barrier Function in Non-IBD Disease

An improperly functioning epithelial barrier is increasingly implicatedin, e.g., IBDs and mucositis. Moreover, there are numerous otherdiseases that studies have shown are also caused, linked, correlated,and/or exacerbated by, an improperly functioning epithelial barrier.These diseases include: (1) metabolic diseases, including-obesity, type2 diabetes, non-alcoholic steatohepatitis (NASH), non-alcoholic fattyliver disease (NAFLD), liver disorders, and alcoholic steatohepatitis(ASH); (2) celiac disease; (3) necrotizing enterocolitis; (4) irritablebowel syndrome (IBS); (5) enteric infections (e.g. Clostridiumdifficile); (6) other gastro intestinal disorders in general; (7)interstitial cystitis; (8) neurological disorders or cognitive disorders(e.g. Alzheimer's, Parkinson's, multiple sclerosis, and autism); (9)chemotherapy associated steatohepatitis (CASH); and (10) pediatricversions of the aforementioned diseases. See, e.g.: Everard et al.,“Responses of Gut Microbiota and Glucose and Lipid Metabolism toPrebiotics in Genetic Obese and Diet-Induced Leptin-Resistant Mice,”Diabetes, Vol. 60, (November 2011), pgs. 2775-2786; Everard et al.,“Cross-talk between Akkermansia muciniphila and intestinal epitheliumcontrols diet-induced obesity,” PNAS, Vol. 110, No. 22, (May 2013), pgs.9066-9071; Cani et al., “Changes in Gut Microbiota Control MetabolicEndotoxemia-Induced Inflammation in High-Fat Diet-Induced Obesity andDiabetes in Mice,” Diabetes, Vol. 57, (June 2008), pgs. 1470-1481;Delzenne et al., “Targeting gut microbiota in obesity: effects ofprebiotics and probiotics,” Nature Reviews, Vol. 7, (November 2011),pgs. 639-646. Consequently, restoring proper epithelial barrier functionto patients may be critical in resolving the aforementioned diseasestates.

A properly functioning epithelial barrier in the lumen of the alimentarycanal, including the mouth, esophagus, stomach, small intestine, largeintestine, and rectum, is critical in controlling and maintaining themicrobiome within the gastrointestinal tract and alimentary canal. Theecosystem for the microbiome includes the environment, barriers,tissues, mucus, mucin, enzymes, nutrients, food, and communities ofmicroorganism, that reside in the gastrointestinal tract and alimentarycanal. The integrity and permeability of the intestinal mucosal barrierimpacts health in many critical ways.

A loss of integrity of the mucosal barrier in gastro-intestinaldisorders due to changes in mucin secretion may be related to hostimmune changes, luminal microbial factors, or directly acting genetic orenvironmental determinants. Thus, the disequilibrium of the mucousbarrier may be central to the pathogenesis of IBD. Boltin et al., “MucinFunction in Inflammatory Bowel Disease an Update,” J. Clin.Gastroenterol., Vol. 47(2):106-111 (February 2013).

Mucins are the primary constituent of the mucous layer lining the GItract. There are at least 21 mucin (MUC) genes known in the humangenome, encoding either secreted or membrane-bound mucins. Thepredominant mucins in the normal colorectum are MUC1, MUC2, MUC3A,MUC3B, MUC4, MUC13, and MUC17.1 MUC2 is the primary secretory,gel-forming component of intestinal mucus, produced in goblet cells.See, Boltin et al., “Mucin Function in Inflammatory Bowel Disease anUpdate,” J. Clin. Gastroenterol., Vol. 47(2):106-111 (February 2013).Along with additional secreted mucins such as MUC1, 3A, 3B, 4, 13 and17.1, goblet cell secretion of MUC2 forms a protective barrier oncolonic epithelial cells reducing exposure to intestinal contents whichmay damage epithelial cells or prime immune responses.

Inflammatory Mechanisms in IBDs

There is significant evidence showing that certain cytokines areinvolved with IBD. Recent studies have demonstrated that cytokines playa crucial role in the pathogenesis of inflammatory bowel diseases(IBDs), such as Crohn's disease and ulcerative colitis, where theycontrol multiple aspects of the inflammatory response. Markus Neurath,“Cytokines in Inflammatory Bowel Disease,” Nature Reviews Immunology,Vol. 14., 329-342 (2014). In particular, the imbalance betweenpro-inflammatory and anti-inflammatory cytokines that occurs in IBDimpedes the resolution of inflammation and instead leads to diseaseperpetuation and tissue destruction. Id. Recent studies suggest theexistence of a network of regulatory cytokines that has importantimplications for disease progression. Id. Accordingly, experiments wereperformed to study the effects of SG-14 on production and/or secretionof pro-inflammatory and anti-inflammatory cytokines.

Pro-Inflammatory Cytokines

Briefly, pro-inflammatory cytokines are cytokines that are important incell signaling and promote systemic inflammation. They are producedpredominantly by activated macrophages and are involved in theupregulation of inflammatory reactions. Pro-inflammatory cytokines arisefrom genes that code for the translation of small mediator moleculesthat induce a response after upregulation. Interleukin-1 (IL-1), IL6,IL-12, IL-18, IL-23, CD40L, tumor necrosis factor (TNF) such as TNF-α,gamma-interferon (IFN-gamma), granulocyte-macrophage colony stimulatingfactor, and MCP-1 are well characterized as pro-inflammatory cytokines.Inflammation is characterized by an interplay between pro- andanti-inflammatory cytokines.

Reducing the biological activities of pro-inflammatory cytokines can beuseful for the treatment of some diseases. For instance, blocking IL-1or TNF-α has been successful in helping patients with rheumatoidarthritis, inflammatory bowel disease, or graft-vs-host disease. See,Strober W, Fuss I J (May 2011). “Proinflammatory cytokines in thepathogenesis of inflammatory bowel diseases,” Gastroenterology, Vol. 140(6): 1756-67.

Anti-Inflammatory Cytokines

Briefly, anti-inflammatory cytokines are a series of immunoregulatorymolecules that regulate the proinflammatory cytokine response. Thesemolecules thus modulate and help to decrease inflammatory responsestriggered by pro-inflammatory cytokines. Anti-inflammatory cytokinesinclude, e.g., IL4, IL-10, IL-13, IFN-α, and transforming growthfactor-beta (TGF-0) are recognized as anti-inflammatory cytokines.

In some embodiments of the methods taught herein, administration of thepharmaceutical composition comprising a SG-14 protein or variant orfragment thereof is able to bring about reduced production of at leastone pro-inflammatory cytokine (e.g., TNF-α and/or IL-23) by an immunecell in a patient administered the composition. In some embodiments, theadministration is able to bring about an increase in the production ofat least one anti-inflammatory cytokine (e.g., IL-10) by an immune cellin the patient. In some embodiments, the administration is able to bringabout a decrease in the production of at least one anti-inflammatorycytokine (e.g., IL-10) by an immune cell in the patient. In someembodiments, the administration is able to bring about an improvement ofmucin production in epithelial cells and/or epithelial wound healing inthe patient

The dosing regimen used for treatment depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment. The dose will vary from patient to patient, dependingupon the nature and severity of disease, the patient's weight, specialdiets then being followed by a patient, concurrent medication, and otherfactors which those skilled in the art will recognize.

Generally, dosage levels of therapeutic protein between 0.0001 to 10mg/kg of body weight daily are administered to the patient, e.g.,patients suffering from inflammatory bowel disease. The dosage rangewill generally be about 0.5 mg to 100.0 g per patient per day, which maybe administered in single or multiple doses.

In some aspects, the dosage range will be about 0.5 mg to 10 g perpatient per day, or 0.5 mg to 9 g per patient per day, or 0.5 mg to 8 gper patient per day, or 0.5 mg to 7 g per patient per day, or 0.5 mg to6 g per patient per day, or 0.5 mg to 5 g per patient per day, or 0.5 mgto 4 g per patient per day, or 0.5 mg to 3 g per patient per day, or 0.5mg to 2 g per patient per day, or 0.5 mg to 1 g per patient per day.

In some aspects, the dosage range will be about 0.5 mg to 900 mg perpatient per day, or 0.5 mg to 800 mg per patient per day, or 0.5 mg to700 mg per patient per day, or 0.5 mg to 600 mg per patient per day, or0.5 mg to 500 mg per patient per day, or 0.5 mg to 400 mg per patientper day, or 0.5 mg to 300 mg per patient per day, or 0.5 mg to 200 mgper patient per day, or 0.5 mg to 100 mg per patient per day, or 0.5 mgto 50 mg per patient per day, or 0.5 mg to 40 mg per patient per day, or0.5 mg to 30 mg per patient per day, or 0.5 mg to 20 mg per patient perday, or 0.5 mg to 10 mg per patient per day, or 0.5 mg to 1 mg perpatient per day.

Combination Therapies Comprising Therapeutic Proteins

The pharmaceutical compositions taught herein comprising a therapeuticprotein may be combined with other treatment therapies and/orpharmaceutical compositions. For example, a patient suffering from aninflammatory bowel disease, may already be taking a pharmaceuticalprescribed by their doctor to treat the condition. In embodiments, thepharmaceutical compositions taught herein, are able to be administeredin conjunction with the patient's existing medicines.

For example, the therapeutic proteins taught herein may be combined withone or more of: an anti-diarrheal, a 5-aminosalicylic acid compound, ananti-inflammatory agent, an antibiotic, an antibody (e.g., antibodiestargeting an inflammatory cytokine, e.g., antibodies targeting ananti-cytokine agent such as anti-TNF-α, (e.g., adalimumab, certolizumabpegol, golimumab, infliximab, V565) or anti-IL-12/IL-23 (e.g.,ustekinumab, risankizumab, brazikumab, ustekinumab), a JAK inhibitor(e.g., tofacitinib, PF06700841, PF06651600, filgotinib, upadacitinib),an anti-integrin agent (e.g., vedolizumab, etrolizumab), a SiP inhibitor(e.g., etrasimod, ozanimod, amiselimod), a recombinant cell-based agent(e.g., Cx601), a steroid, a corticosteroid, an immunosuppressant (e.g.,azathioprine and mercaptopurine), vitamins, and/or specialized diet.

Cancer patients undergoing chemotherapy or radiation therapy andsuffering from or at risk of developing mucositis, e.g., oral mucositis,may be administered a pharmaceutical composition according to thepresent disclosure in combination with an agent used to treat mucositissuch as oral mucositis. In some embodiments, a method of treatmentcomprises administering to a patient suffering from mucositis acombination of a pharmaceutical composition comprising SG-14 or avariant or fragment thereof and one or more second therapeutic agentsselected from the group consisting of amifostine, benzocaine,benzydamine, ranitidine, omeprazole, capsaicin, glutamine, prostaglandinE2, Vitamin E, sucralfate, and allopurinol.

In some embodiments of the methods herein, the second therapeutic agentis administered in conjunction with the SG-14 protein described herein,either simultaneously or sequentially. In some embodiments, the proteinand the second agent act synergistically for treatment or prevention ofthe disease, or condition, or symptom. In other embodiments, the proteinand the second agent act additively for treatment or prevention of thedisease, or condition, or symptom.

Pharmaceutical Compositions Comprising the SG-14 Therapeutic Protein

Pharmaceutical compositions are provided herein which comprise a SG-14protein, variant or fragment thereof according to the present disclosureor pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient. In some embodiments, the pharmaceuticalcomposition is formulated for administration to the gastrointestinallumen, including the mouth, esophagus, small intestine, large intestine,rectum and/or anus.

In some embodiments, the composition comprises one or more othersubstances which are associated with the source of the protein, forexample, cellular components from a production host cell, or substanceassociated with chemical synthesis of the protein. In other embodiments,the pharmaceutical composition is formulated to include one or moresecond active agents as described herein. Moreover, the composition maycomprise ingredients that preserve the structural and/or functionalactivity of the active agent(s) or of the composition itself. Suchingredients include but are not limited to antioxidants and variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

The terms “pharmaceutical” or pharmaceutically acceptable” referscompositions that do not or preferably do not produce an adverse,allergic, or other untoward reaction when administered to an animal,such as, for example, a human, as appropriate. The preparation of apharmaceutical composition or additional active ingredient will be knownto those of skill in the art in light of the present disclosure, asexemplified by Remington's Pharmaceutical Sciences, 18^(th) Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by the FDA Office of Biological Standards.

The pharmaceutical compositions of the disclosure are formulatedaccording to the intended route of administration and whether it is tobe administered, e.g., in solid, liquid or aerosol form. In a preferredembodiment, the composition can be administered rectally, but may alsobe administered topically, by injection, by infusion, orally,intrathecally, intranasally, subcutaneously, mucosally, localizedperfusion bathing target cells directly, via a catheter, via a lavage,or by other method or any combination of the foregoing as would be knownto one of ordinary skill in the art. Liquid formulations comprising atherapeutically effective amount of the protein can be administeredrectally by enema, catheter, use of a bulb syringe. A suppository is anexample of a solid dosage form formulated for rectal delivery. Ingeneral, for suppositories, traditional carriers may include, forexample, polyalkylene glycols, triglycerides or combinations thereof. Incertain embodiments, suppositories may be formed from mixturescontaining, for example, the active ingredient in the range of about0.5% to about 10%, and or about 1% to about 2%. Injectable liquidcompositions are typically based upon injectable sterile saline orphosphate-buffered saline or other injectable carriers known in the art.Other liquid compositions include suspensions and emulsions. Solidcompositions such as for oral administration may be in the form oftablets, pills, capsules (e.g., hard or soft-shelled gelatin capsules),buccal compositions, troches, elixirs, suspensions, syrups, wafers, orcombinations thereof. The active agent in such liquid and solidcompositions, i.e., a protein as described herein, is typically acomponent, being about 0.05% to 10% by weight, with the remainder beingthe injectable carrier and the like.

The pharmaceutical composition may be formulated as a controlled orsustained release composition which provide release of the activeagent(s) including the therapeutic protein of the present disclosureover an extended period of time, e.g., over 30-60 minutes, or over 1-10hours, 2-8 hours, 8-24 hours, etc. Alternatively or additionally, thecomposition is formulated for release to a specific site in the hostbody. For example, the composition may have an enteric coating toprevent release of the active agent(s) in an acidic environment such asthe stomach, allowing release only in the more neutral or basicenvironment of the small intestine, colon or rectum. Alternatively oradditionally, the composition may be formulated to provide delayedrelease in the mouth, small intestine or large intestine.

Each of the above-described formulations may contain at least onepharmaceutically acceptable excipient or carrier, depending up theintended route of administration, e.g., a solid for rectaladministration or liquid for intravenous or parenteral administration oradministration via cannula. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences. 18^(th) Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference).

The pharmaceutical compositions for administration can be present inunit dosage forms to facilitate accurate dosing. Typical unit dosageforms include prefilled, premeasured ampules or syringes of the liquidcompositions or suppositories, pills, tablets, capsules or the like inthe case of solid compositions. In some embodiments of suchcompositions, the active agent, i.e., a protein as described herein, maybe a component (about 0.1 to 50 wt/wt %, 1 to 40 wt/wt %, 0.1 to 1 wt/wt%, or 1 to 10 wt/wt %) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

The actual dosage amount in a unit dosage form of the present disclosureadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

Protein Expression Systems and Protein Production

Provided herein are compositions and methods for producing isolatedproteins of the present disclosure as well as expression vectors whichcontain polynucleotide sequence encoding the proteins and host cellswhich harbor the expression vectors.

The proteins of the present disclosure can be prepared by routinerecombinant methods, e.g., culturing cells transformed or transfectedwith an expression vector containing a nucleic acid encoding the SG-14therapeutic protein, variant or fragment thereof. Host cells comprisingany such vector are also provided. Host cells can be prokaryotic oreukaryotic and examples of host cells include E. coli, yeast, ormammalian cells. A method for producing any of the herein describedproteins is further provided and comprises culturing host cells underconditions suitable for expression of the desired protein and recoveringthe desired protein from the cell culture. The recovered protein canthen be isolated and/or purified for use in in vitro and in vivomethods, as well as for formulation into a pharmaceutically acceptablecomposition. In some embodiments, the protein is expressed in aprokaryotic cell such as E. coli and the isolation and purification ofthe protein includes step to reduce endotoxin to levels acceptable fortherapeutic use in humans or other animals.

Expression Vectors

Provided herein are expression vectors which comprise a polynucleotidesequence which encodes a protein of the present disclosure or a variantand/or fragment thereof. Polynucleotide sequences encoding the proteinsof the disclosure can be obtained using standard recombinant techniques.Desired encoding polynucleotide sequences may be amplified from thegenomic DNA of the source bacterium, i.e., E. eligens. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologous(exogenous) polynucleotides in a host cell. Many vectors that areavailable and known in the art can be used for the purpose of thepresent disclosure. Selection of an appropriate vector will dependmainly on the size of the nucleic acids to be inserted into the vectorand the particular host cell to be transformed with the vector. Eachvector contains various components, depending on its function(amplification or expression of heterologous polynucleotide, or both)and its compatibility with the particular host cell in which it resides.The vector components generally include, but are not limited to: anorigin of replication, a selection marker gene, a promoter, a ribosomebinding site (RBS), a signal sequence, the heterologous nucleic acidinsert and a transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using a pBR322, pUC, pET or pGEX vector, a plasmidderived from an E. coli species. Such vectors contain genes encodingampicillin (Amp) and tetracycline (Tet) resistance and thus provideseasy means for identifying transformed cells. These vectors as well astheir derivatives or other microbial plasmids or bacteriophage may alsocontain, or be modified to contain, promoters which can be used by themicrobial organism for expression of endogenous proteins.

An expression vector of the present disclosure may comprise a promoter,an untranslated regulatory sequence located upstream (5′) and operablylinked to a protein-encoding nucleotide sequence such that the promoterregulated transcription of that coding sequence. Prokaryotic promoterstypically fall into two classes, inducible and constitutive. Aninducible promoter is a promoter that initiates increased levels oftranscription of the encoding polynucleotide under its control inresponse to changes in the culture condition, e.g., the presence orabsence of a nutrient or a change in temperature. A large number ofpromoters recognized by a variety of potential host cells are well knownand a skilled artisan can choose the promoter according to desiredexpression levels. Promoters suitable for use with prokaryotic hostsinclude E. coli promoters such as lac, trp, tac, trc and ara, viralpromoters recognized by E. coli such as lambda and T5 promoters, and theT7 and T71ac promoters derived from T7 bacteriophage. A host cellharboring a vector comprising a T7 promoter, e.g., is engineered toexpress a 17 polymerase. Such host cells include E. coli BL21(DE3),Lemo21(DE3), and NiCo21(DE3) cells. In some embodiments, the promoter isan inducible promoter which is under the control of chemical orenvironmental factors.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S-transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like.

Suitable vectors for expression in both prokaryotic and eukaryotic hostcells are known in the art and some are further described herein.

Vectors of the present disclosure may further comprise a signal sequencewhich allows the translated recombinant protein to be recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process the signalsequences native to the heterologous polypeptides, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group consisting of the alkaline phosphatase, penicillinase,Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB,OmpA and MBP. Well-known signal sequences for use in eukaryoticexpression systems include but are not limited to interleukin-2, CD5,the Immunoglobulin Kappa light chain, trypsinogen, serum albumin, andprolactin.

The SG-14 proteins or variants or fragments thereof as described hereincan be expressed as a fusion protein or polypeptide. Commonly usedfusion partners include but are not limited to human serum albumin andthe crystallizable fragment, or constant domain of IgG, Fc. Thehistidine tag or FLAG tag can also be used to simplify purification ofrecombinant protein from the expression media or recombinant celllysate. The fusion partners can be fused to the N- and/or C-terminus ofthe protein of interest.

Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained for example through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials. Cell types available for vectorreplication and/or expression include, but are not limited to, bacteria,such as E. coli (e.g., E. coli strain RR1, E. coli LE392, E. coli B, E.coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-,prototrophic, ATCC No. 273325), DH5α, JM109, and KC8, bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium. Serratia marcescens, various Pseudomonas species, as wellas a number of commercially available bacterial hosts such as SURE®Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Incertain embodiments, bacterial cells such as E. coli are particularlycontemplated as host cells.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Additional eukaryotic host cells include yeasts(e.g., Pichia pastoris and Saccharomyces cerevisiae) and cells derivedfrom insects (e.g., Spodoptera frugiperda or Trichoplusia ni). Many hostcells from various cell types and organisms are available and would beknown to one of skill in the art. Similarly, a viral vector may be usedin conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector. The selection of the appropriate host cell is deemed to bewithin the skill in the art.

Methods are well known for introducing recombinant DNA, i.e., anexpression vector, into a host cell so that the DNA is replicable,either as an extrachromosomal element or as a chromosomal integrant,thereby generating a host cell which harbors the expression vector ofinterest. Methods of transfection are known to the ordinarily skilledartisan, for example, by CaPO₄ and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact, 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). Other methods forintroducing DNA into cells include nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orintroduction using polycations, e.g., polybrene, polyomithine. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology. 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Accordingly, provided herein is a recombinant vector or expressionvector as described above and comprising a polynucleotide which encodesa SG-14 therapeutic protein sequence of interest (e.g., SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7 or variant and/or fragment thereof asdescribed herein). The polynucleotide can be, for example, any one ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or a variant or afragment thereof. Moreover, the present disclosure teaches a host cellharboring the vector. The host cell can be a eukaryotic or prokaryoticcell as detailed above. In a preferred embodiment, the host cell is aprokaryotic cell. In a further preferred embodiment, the host cell is E.coli. The recombinant expression vector may be constructed to expressthe SG-14 protein with, e.g, an affinity tag at the N-terminus and/orthe C-terminus. The affinity tag can be linked to the SG-14 protein viaa short amino acid sequence which contains a protease cleavage site.Accordingly, after cleavage of the expressed protein with the protease,the SG-14 protein may contain exogenous amino acids at the N- orC-terminus of the therapeutic protein without affecting functionalactivity of the protein.

In some embodiments, the polynucleotide encoding the protein of interestis codon-optimized. A codon optimization algorithm is applied to apolynucleotide sequence encoding a protein in order to choose anappropriate codon for a given amino acid based on the expression host'scodon usage bias. Many codon optimization algorithms also take intoaccount other factors such as mRNA structure, host GC content, ribosomalentry sites. Some examples of codon optimization algorithms and genesynthesis service providers are: AUTM: www.atum.bio/services/genegps;GenScript: www.genscript.com/codon-opt.html; ThermoFisher:www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html;and Integrated DNA Technologies: www.idtdna.com/CodonOpt. The nucleotidesequence is then synthesized and cloned into an appropriate expressionvector.

Methods to Produce the Protein

Methods are provided for producing the proteins described herein but arewell known to the ordinarily skilled artisan. Host cells transformed ortransfected with expression or cloning vectors described herein forprotein production are cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting and/or maintainingtransformants, and/or expressing the genes encoding the desired proteinsequences. The culture conditions, such as media, temperature, pH andthe like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.(IRL Press, 1991) and Molecular Cloning: A Laboratory Manual (Sambrook,et al., 1989, Cold Spring Harbor Laboratory Press).

Generally, “purified” will refer to a specific protein composition thathas been subjected to fractionation to remove non-proteinaceouscomponents and various other proteins, polypeptides, or peptides, andwhich composition substantially retains its activity, as may beassessed, for example, by the protein assays, as described herein below,or as would be known to one of ordinary skill in the art for the desiredprotein, polypeptide or peptide.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific protein, polypeptide, or peptide formsthe major component of the composition, such as constituting about 50%of the proteins in the composition or more. In preferred embodiments, asubstantially purified protein will constitute more than 60%, 70%, 80%,90%, 95%, 99% or even more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present disclosure, means that the peptide, polypeptideor protein has a level of purity where the peptide, polypeptide orprotein is substantially free from other proteins and biologicalcomponents. For example, a purified peptide, polypeptide or protein willoften be sufficiently free of other protein components so thatdegradative sequencing may be performed successfully.

Although preferred for use in certain embodiments, there is no generalrequirement that the protein, polypeptide, or peptide always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified protein, polypeptide or peptide, which arenonetheless enriched in the desired protein compositions, relative tothe natural state, will have utility in certain embodiments.

Various methods for quantifying the degree of purification of proteins,polypeptides, or peptides will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. As the present disclosure provides DNA sequences for the specificproteins, any fusion protein purification method can now be practiced.This is exemplified by the generation of a specific protein-glutathioneS-transferase fusion protein, expression in E. coli, and isolation tohomogeneity using affinity chromatography on glutathione-agarose or thegeneration of a poly-histidine tag on the N- or C-terminus of theprotein, and subsequent purification using Ni-affinity chromatography.However, given many DNA and proteins are known, or may be identified andamplified using the methods described herein, any purification methodcan now be employed.

In other embodiments, a preparation enriched with the peptides may beused instead of a purified preparation. In this document, wheneverpurified is used, enriched may be used also. A preparation may not onlybe enriched by methods of purification, but also by the over-expressionor over-production of the peptide by bacteria when compared towild-type. This can be accomplished using recombinant methods, or byselecting conditions which will induce the expression of the peptidefrom the wild type cells.

Recombinantly expressed polypeptides of the present disclosure can berecovered from culture medium or from host cell lysates. The suitablepurification procedures include, for example, by fractionation on anion-exchange (anion or cation) column; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration or size exclusion chromatograph (SEC) using, for example,Sephadex G-75; and metal chelating columns to bind epitope-tagged formsof a polypeptide of the present disclosure. Various methods of proteinpurification can be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularpolypeptide produced.

Alternative methods, which are well known in the art, can be employed toprepare a polypeptide of the present invention. For example, a sequenceencoding a polypeptide or portion thereof, can be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., 1969, Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif.; Merrifield. J. 1963, Am. Chem. Soc., 85:2149-2154. Invitro protein synthesis can be performed using manual techniques or byautomation. Automated synthesis can be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of a polypeptide of thepresent invention or portion thereof can be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length polypeptide or portion thereof.

In some embodiments, the disclosure provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence and the polynucleotidesencoding the chimeric molecules. Examples of such chimeric moleculesinclude, but are not limited to, any of the herein describedpolypeptides fused to an epitope tag sequence, an Fc region of animmunoglobulin.

Recombinant Bacterial Delivery Systems

The present disclosure contemplates utilizing delivery systems outsideof the traditional pharmaceutical formulations that comprise a purifiedprotein. In some embodiments, the disclosure utilizes recombinantbacterial delivery systems, phage-mediated delivery systems,chitosan-DNA complexes, or AAV delivery systems.

One particular recombinant bacterial delivery system is based uponLactococcus lactis. Essentially, one may clone the gene encoding thetherapeutic protein (e.g. SEQ ID NO:3) into an expression vector, andthen transform the vector into L. lactis. Subsequently, one may thenadminister the L. lactis to a patient. See, e.g. Bratt, et al., “A phase1 trial with transgenic bacteria expressing interleukin-10 in Crohn'sdisease,” Clinical Gastroenterology and Hepatology, 2006, Vol. 4, pgs.754-759 (“We treated Crohn's disease patients with genetically modifiedLactococcus lactis (LL-Thy12) in which the thymidylate synthase gene wasreplaced with a synthetic sequence encoding mature humaninterleukin-10.”); Shigemori, et al., “Oral delivery of Lactococcuslactis that secretes bioactive heme oxygenase-1 alleviates developmentof acute colitis in mice,” Microbial Cell Factories, 2015, Vol. 14:189(“Mucosal delivery of therapeutic proteins using genetically modifiedstrains of lactic acid bacteria (gmLAB) is being investigated as a newtherapeutic strategy.”); Steidler, et al., “Treatment of murine colitisby Lactococcus lactis secreting interleukin-10,” Science, 2000, Vol.289, pgs. 1352-1355 (“The cytokine interleukin-10 (IL-10) has shownpromise in clinical trials for treatment of inflammatory bowel disease(IBD). Using two mouse models, we show that the therapeutic dose ofIL-10 can be reduced by localized delivery of a bacterium geneticallyengineered to secrete the cytokine. Intragastric administration ofIL-10Ðsecreting Lactococcus lactis caused a 50% reduction in colitis inmice treated with dextran sulfate sodium and prevented the onset ofcolitis in IL-102/2 mice. This approach may lead to better methods forcost effective and long-term management of IBD in humans.”); Hanniflfy,et al., “Mucosal delivery of a pneumococcal vaccine using Lactococcuslactis affords protection against respiratory infection,” Joumal ofInfectious Diseases, 2007, Vol. 195, pgs. 185-193 (“Here, we evaluatedLactococcus lactis intracellularly producing the pneumococcal surfaceprotein A (PspA) as a mucosal vaccine in conferring protection againstpneumococcal disease.”); and Vandenbroucke, et al., “Active delivery oftrefoil factors by genetically modified Lactococcus lactis prevents andheals acute colitis in mice,” Gastroenterology, 2004, Vol. 127, pgs.502-513 (“We have positively evaluated a new therapeutic approach foracute and chronic colitis that involves in situ secretion of murine TFFby orally administered L. lactis. This novel approach may lead toeffective management of acute and chronic colitis and epithelial damagein humans.”).

In another embodiment, a “synthetic bacterium” may be used to deliver anSG-14 protein or variant or fragment thereof wherein a probioticbacterium is engineered to express the SG-14 therapeutic protein (see,e.g., Durrer and Allen, 2017, PLoS One, 12:e0176286).

Phages have been genetically engineered to deliver specific DNA payloadsor to alter host specificity. Transfer methods, such as phages,plasmids, and transposons, can be used to deliver and circulateengineered DNA sequences to microbial communities, via processes such astransduction, transformation, and conjugation. For purposes of thepresent disclosure, it is sufficient to understand that an engineeredphage could be one possible delivery system for a protein of thedisclosure, by incorporating the nucleic acid encoding said protein intothe phage and utilizing the phage to deliver the nucleic acid to a hostmicrobe that would then produce the protein after having the phagedeliver the nucleic acid into its genome.

Similar to the aforementioned engineered phage approach, one couldutilize a transposon delivery system to incorporate nucleic acidsencoding a therapeutic protein into a host microbe that is resident in apatient's microbiome. See, Sheth, et al., “Manipulating bacterialcommunities by in situ microbiome engineering,” Trends in Genetics,2016, Vol. 32, Issue 4, pgs. 189-200.

The following examples are intended to illustrate, but not limit, thedisclosure.

EXAMPLES

The following experiments utilize a robust mixture of in vitroexperiments combined with in vivo models of IBD to demonstrate thetherapeutic ability of the taught proteins and methods.

Example 1 Expression of SG-14

For experiments described in the examples below, a polynucleotidecomprising a sequence encoding residues 1-631 of SEQ ID NO:3 (SG-14) wasobtained by PCR amplification of genomic DNA obtained from Eubacteriumeligens (C15-B4; DSM 3376 type strain; See, e.g., Holdeman, L. V.,Moore, W. E. C. (1974) New genus, Coprococcus, twelve new species, andamended descriptions of four previously described species of bacteriafrom human feces. Int J Syst Bacteriol 24, 260-277. The encodingpolynucleotide was then subcloned into an inducible expression vectorand used to transform E. coli BL21(DE3) cells for expression andpurification of SG-14 as detailed below, using culturing andpurification methods which are routine in the art.

Expression and purification of proteins for use in various experimentspertaining to the present disclosure and for the following examples isdescribed here. Expression was achieved using a pGEX vector system whichis designed for inducible, high-level intracellular expression of genesor gene fragments. Expression in E. coli yields tagged proteins with theGST moiety at the amino terminus and the protein of interest at thecarboxyl terminus. The vector has a tac promoter for chemicallyinducible, high-level expression and an internal laq 1 gene for use inany E. coli host.

The polynucleotide comprising a nucleotide sequence (PCR-amplified fromE. eligens DSM 3376 and encoding residues 1 to 631 of SEQ ID NO:3) wasinserted into the BamHI and NotI sties of the multiple-cloning site of apGEX-6P-1 vector (GE Healthcare Life Science, Pittsburgh, PA) to expressSG-14 as a GST fusion protein which was then cleaved at the Precisionprotease site, whereby an exogenous GPLG sequence is present at theN-terminus and PHRD is present at the C-terminus of the cleaved andpurified protein. BL21(DE) E. coli cells were transformed with theexpression construct. BL21(DE3) transformants were grown in 10 L LB with50 pg/ml carbenicillin at 37° C. When cultures reached a density of0.8ODwo, they were chilled to 16° C. and expression was induced with 1 mMIPTG at 16° C. for 16 h. Cells were harvested and lysed by sonication,and a soluble lysate was applied to a GSTrap column. Bound protein waswashed with HEPES buffer and then purified tag-free SG-14 was eluted byadding HRV3C protease to cleave the protein C-terminal to the GST-tag.Eluted fractions containing protein as determined by SDS-PAGE andCoomasie Brilliant Blue staining were identified and pooled, thenapplied to a HiTrap Q HP anion exchange column then to a HiLoad Superdex200 26/60 preparative size exclusion column (SEC) to obtain a finalpreparation.

In an alternative method of expression and purification with the sameexpression construct, BL21 transformants were grown in LB and 100 pg/mlcarbenicillin and 1 pg/ml chloramphenicol at 30° C. Expression wasinduced when a culture density of 0.6 OD₆₀₀ was reached, with 0.4 mMIPTG for 4 h. Cells were harvested by centrifugation then lysed bysonication, and a soluble lysate was applied to a GST-resin column.Bound protein was washed with PBS and then purified tag-free SG-14 waseluted by adding PreScission Protease to cleave the protein C-terminalto the GST-tag.

Purified proteins were quantified by densitometry using bovine serumalbumin as a reference following SDS-PAGE and Coomassie Brilliant Bluestaining. Endotoxin levels were measured with Endosafe® nexgen-MCS™(Charles River, Wilmington, MA) according to the manufacturer'sinstructions. Endotoxin levels of proteins used for the assays describedherein were lower than 1 EU/mg.

Example 2 Effect of SG-14 on Restoration of Epithelial Barrier IntegrityFollowing Inflammation Induced Disruption

The following experiment demonstrates the therapeutic ability of a SG-14protein or variant or fragment thereof as disclosed herein to restoregastrointestinal epithelial barrier integrity. The experiment istherefore a demonstration of the functional utility of the therapeuticprotein SG-14 to treat a gastrointestinal inflammatory disorder ordisease involving impaired epithelial barrier integrity/function.

Assays were performed as described below in trans-well plates whereco-cultures of multiple cell types were performed utilizing a permeablemembrane to separate cells. In the apical (top) chamber, human colonicepithelial cells, consisting of a mixture of enterocytes and gobletcells, were cultured until cells obtained tight junction formation andbarrier function capacity as assessed by measurement of trans-epithelialelectrical resistance (TEER). In the basolateral chamber, monocytes werecultured separately. Epithelial cells were primed with inflammatorycytokines. The assays measured the effect of a therapeutic protein,i.e., SG-14, on epithelial barrier function, muc2 gene expression, andproduction of cytokines.

Cell culture. The HCT8 human enterocyte cell line (ATCC Cat. No.CCL-244) was maintained in RPMI-1640 medium supplemented with 10% fetalbovine serum, 100 IU/ml penicillin, 100 pg/ml streptomycin, 10 pg/mlgentamicin and 0.25 pg/ml amphotericin (cRPMI). HT29-MTX human gobletcells (Sigma-Aldrich (St. Louis, MO; Cat. No. 12040401) were maintainedin DMEM medium with 10% fetal bovine serum, 100 IU/ml penicillin, 100pg/ml streptomycin, 10 pg/ml gentamicin and 0.25 pg/ml amphotericin(cDMEM). Epithelial cells were passaged by trypsinization and were usedbetween 5 and 15 passages following thawing from liquid nitrogen stocks.U937 monocytes (ATCC Cat. No. 700928) were maintained in cRPMI medium asa suspension culture, and split by dilution as needed to maintain cellsbetween 5×10⁵ and 2×10⁶ cells/ml. U937 cells were used up to passage 18following thawing from liquid nitrogen stocks.

Epithelial cell culture. A mixture of HCT8 enterocytes and HT29-MTXgoblet cells were plated at a 9:1 ratio, respectively, in the apicalchamber of the transwell plate as described previously (Berget et al.,2017, Int J Mol Sci, 18:1573; Beduneau et al., 2014, Eur J PharmBiopharm, 87:290-298). A total of 10¹ cells were plated in each well(9×10⁴ HCT8 cells and 1×10⁴ HT29-MTX cells per well). Epithelial cellswere trypsinized from culture flasks and viable cells determined bytrypan blue counting. The correct volumes of each cell type werecombined in a single tube and centrifuged. The cell pellet wasresuspended in cRPMI and added to the apical chamber of the transwellplate. Cells were cultured for 8 to 10 days at 37° C.+5% CO₂, and mediawas changed every 2 days.

Monocyte culture. On day 6 of epithelial cell culture 2×10⁵ cells/wellU937 monocytes were plated into a 96 well receiver plate. Cells werecultured at 37° C.+5% CO₂ and media was changed every 24 hours for fourdays.

Co-culture assay. Following 8-10 days of culture the transwell platecontaining enterocytes were treated with 10 ng/ml IFN-γ added to thebasolateral chamber of the transwell plate for 24 hours at 37° C.+5%CO₂. After 24 hours fresh cRPMI was added to the epithelial cell cultureplate. Trans-epithelial electrical resistance (TEER) readings weremeasured after the IFN-γ treatment and were used as the pre-treatmentTEER values. SG-14 was then added to the apical chamber of the transwellplate at a final concentration of 1 pg/ml (40 nM). The myosin lightchain kinase (MLCK) inhibitor peptide 18 (BioTechne, Minneapolis, MN)was used at 50 nM as a positive control to prevent inflammation inducedbarrier disruption (Zolotarevskky et al., 202, Gastroenterology,123:163-172). The bacterially derived molecule staurosporine was used at100 nM as a negative control to induce apoptosis and exacerbate barrierdisruption (Antonsson and Persson, 2009, Anticancer Res, 29:2893-2898).Compounds were incubated on enterocytes for 1 hour or 6 hours. Followingpre-incubation with test compounds the transwell insert containing theenterocytes was transferred on top of the receiver plate containing U937monocytes. Heat killed E. coli (HK E. coli) (bacteria heated to 80° C.for 40 minutes) was then added to both the apical and basolateralchambers and a multiplicity of infection (MOI) of 10. Transwell plateswere incubated at 37° C.+5% CO₂ for 24 hours and a post treatment TEERmeasurement was made. The TEER assays were performed with recombinantlyexpressed mature (lacking a signal peptide) SG-14 protein such as thatdescribed in Example 1 above.

Data analysis. Raw electrical resistance values in ohms (0) wereconverted to ohms per square centimeter (Ωcm²) based on the surface areaof the transwell insert (0.143 cm²). To adjust for differentialresistances developing over 10 days of culture, individual well posttreatment Ωcm² readings were normalized to pre-treatment A2 cm²readings. Normalized Ωcm² values were then expressed as a percent changefrom the mean 0 cm² values of untreated samples.

SG-14 protein was added 1 hour (FIG. 1A) or 6 hours (FIG. 1B) prior toexposure of both epithelial cells and monocytes to heat killedEscherichia coli (HK E. coli), inducing monocytes to produceinflammatory mediators resulting in disruption of the epithelialmonolayer as indicated by a reduction in TEER. A myosin light chainkinase (MLCK) inhibitor was utilized as a control compound, which hasbeen shown to prevent barrier disruption and/or reverse barrier losstriggered by the antibacterial immune response. Staurosporine was usedas a control compound that caused epithelial cell apoptosis and/ordeath, thus resulting in a drastic decrease in TEER, which indicatesdisruption and/or loss of epithelial cell barrier integrity/function. InFIG. 1A, TEER of epithelial cell barrier increased from 70.0% only whenepithelial cells were exposed to heat killed E. coli to 72.2% whenincubating epithelial cells with SG-14 for 30 minutes before exposing toHK E. coli. In FIG. 1B, epithelial cells pre-incubated with SG-14 for 6hours before HK E. coli exposure showed increase to 78.7% in TEER from a71.4% without SG-14 treatment. The graphs in FIGS. 1A-1B represent datapooled from two individual experiments (n=6).

Example 3 Effect of SG-14 on TNF-α and IL-23 Production in a TEER Assay

The following experiment demonstrates the therapeutic ability of a SG-14protein or variant thereof as disclosed herein to reduce immuneactivation as measured by cytokine production. The experiment therebydemonstrates potential functional utility of the therapeutic protein totreat a gastrointestinal inflammatory disease, or disease involvingimpaired epithelial barrier integrity/function, where modulation ofcytokine levels would affect the disease state in a host.

Production of the pro-inflammatory cytokines TNF-α and IL-23 bymonocytes was measured in the tissue culture supernatant from thebasolateral chamber of the co-culture TEER assay performed in Example 2.Following TEER readings, the supernatants were centrifuged at 10,000 gfor 5 minutes at 4° C. to remove cell debris. Luminex analysis wasperformed according to the manufacturer's instructions (Magpixinstrument and xPonent software version 4.2; Luminex Corporation(Austin, TX)). Luminex analysis was performed to determine the pg/mlconcentration of TNF-α and IL-23 produced by monocytes. Luminex analysisutilizes a bead-based system for quantification of multiple cytokinesfrom a single sample. Like an ELISA, Luminex beads are coated with acapture antibody, and incubation with samples allows the target to bindto the capture antibody. Beads are washed and incubated with afluorescently labeled detection antibody for quantification of boundtarget. Cytometric analysis is used to differentiate bead populations,which are loaded with differential fluorescent dyes, and to quantifycytokine levels by measuring detection antibody signal.

TNF-α production in untreated cells and cells pre-incubated with SG-14for 6 hours prior to HK E. coli treatment were normalized to pg/mlconcentrations elicited by HK E. coli which was set to 1.0.Pre-incubation with SG-14 significantly reduced TNF-α production to0.77. Results are shown in FIG. 2A. Pre-incubation with SG-14 increasedIL-23 production slightly to 1.1 (p=0.80). Results are shown in FIG. 2B.The graphs in FIGS. 2A and 2B represent data pooled from two individualexperiments (n=6).

Example 4

Effect of SG-14 on IL-10 production induced by heat killed Escherichiacol

The following experiment measures effects of SG-14 administration onIL-10 production in a TEER assay.

IL-10 production was measured in tissue culture supernatant from thebasolateral chamber of the co-culture TEER assay described in Example 2containing monocytes. Luminex analysis was performed to determine thepg/ml concentration of IL-10 produced by the monocytes. IL-10 productionin untreated cells and cells pre-incubated with SG-14 for 6 hours priorto HK E. coli treatment were normalized to pg/ml concentrations elicitedby HK E. coli which was set to 1.0. Pre-incubation with SG-14 increasedIL-10 production to 1.21. Results are shown in FIG. 3 . The graph inFIG. 3 represents data pooled from two individual experiments (n=6).

Example 5

Effects of SG-14 on Mucin Expression Following Stimulation with HeatKilled Escherichia coli

The following experiment measures effects of a SG-14 protein asdisclosed herein to increase mucin expression in gastrointestinaltissue. The experiment is therefore a demonstration of the functionalutility of the therapeutic protein SG-14 to treat a gastrointestinalinflammatory disease, or disease involving impaired epithelial barrierintegrity/function, where increased mucin expression may be beneficial.

Gene expression was measured in the epithelial cell monolayer from theapical chamber of the co-culture TEER assay described in Example 2.Total RNA was isolated from the epithelial cell monolayer, cDNA wassynthesized. qRT-PCR was performed on cDNA generated from the HCT8 &HT29-MTX cells treated with SG-14 (1 pg/ml; 40 nM) for 6 hours prior toaddition of HK E. coli for 24 hours. muc2 gene expression is graphed asmean fold change ±SEM. Statistical analysis was performed by a one-wayANOVA compared to HK E. coli and a Fishers LSD test was used formultiple comparisons.

A slight inhibition of muc2 gene expression was observed in theepithelial cell monolayer with SG-14 pre-treatment for 6 hours prior toHK E. coli stimulation, compared to the epithelial cell monolayerstimulated by HK E. coli without SG-14 pre-treatment (p=0.87). Resultsare shown in FIG. 4 . The graph in FIG. 4 represents data pooled fromtwo independent experiments (n=6).

Example 6 Effects of SG-14 on Epithelial Cell Wound Healing

The following experiment demonstrates the therapeutic ability of aprotein as disclosed herein to increase gastrointestinal epithelial cellwound healing. The experiment is therefore a demonstration of thefunctional utility of the therapeutic protein SG-14 to treat agastrointestinal inflammatory disease, or disease involving impairedepithelial barrier integrity/function, where increased epithelial cellwound healing would be beneficial.

The 96 well Oris Cell Migration assay containing plugs preventing cellattachment in the center of each well was used according to themanufacturer's instructions (Platypus Technologies, Madison, WI).

The migration assay plates were warmed to room temperature prior to useand plugs were removed from 100% confluence wells prior to celladdition. The HCT8 enterocyte and HT29-MTX goblet cell lines were usedat a 9:1 ratio with a total of 5×10⁴ total cells added per well (4.5×10⁴HCT8 cells and 0.5×10⁴ HT29-MTX cells). Cells were incubated at 37°C.+5% CO₂ for 24 hours. Plugs were then removed from all control andsample wells. Control wells included cells treated with the diluentvehicle as the blank, 30 ng/ml epidermal growth factor (EGF) as thepositive control, and 100 nM staurosporine as the negative control, alldiluted in cRPMI. Sample wells contained SG-14 protein at aconcentration of lpg/ml diluted in cRPMI. 100% and 0% wells werecultured in cRPMI. Treatments were added to cells and incubated at 37°C.+5% CO₂ for 48 hours. Prior to staining for viable cells, plugs wereremoved from the 0% wells. Treatment media was removed and cells werewashed in PBS containing 0.9 mM CaCl₂) and 0.5 mM MgCl₂. The greenfluorescent viability dye Calcenin AM was added to all wells at aconcentration of 0.5 pg/ml in PBS containing 0.9 mM CaCl₂) and 0.5 mMMgCl₂, incubated for 30 min at 37° C.+5% CO₂, the dye was removed andcells were washed in PBS containing 0.9 mM CaCl₂) and 0.5 mM MgCl₂ andfluorescence was measured. Relative fluorescent values from 100% wellswhere plugs were removed prior to cell plating were set as the maxeffect, and 0% wells where plugs remained in place until immediatelybefore staining were used as the baseline. Samples were normalizedbetween 100% and 0% samples and values expressed as a percent growth.

As shown in FIG. 5 , a significant increase in growth was observed withSG-14. In addition, control compounds modulated wound healing asexpected with EGF increasing proliferation, and staurosporinesuppressing cell proliferation. The graph in FIG. 5 represents datapooled from 4 replicate experiments (n=18).

Example 7 SG-14 Demonstrates Therapeutic Activity in a DSS Model ofInflammatory Bowel Disease

Example 7 demonstrates the ability of a protein as disclosed herein totreat inflammatory bowel disease in an in vivo model. The experiment istherefore a demonstration that the aforementioned in vitro models, whichdescribed important functional and possible mechanistic modes of action,will translate into an in vivo model system of inflammatory boweldisease. Specifically, the mice in Example 7 were treated with dextransodium sulfate (DSS), a chemical known to induce intestinal epithelialdamage and thereby reduce intestinal barrier integrity and function. DSSmice are well-accepted models of colitis. In Example 7, mice weretreated with SG-14 protein approximately concurrent with (6 hours priorto) administration of DSS. The mice were then assessed for effects ofSG-14 on clinically relevant markers of improved health.

The graphs presented in Example 7 represent data pooled from oneexperiment using 10 mice (n=10). The SG-14 protein used in theseexperiments was the mature protein (no signal peptide), expressed asdescribed in Example 1 above.

Eight-week old C57BL/6 mice were housed 5 animals were cage and givenfood and water ad libitum for 7 days. Following the 7-day acclimationperiod, treatments were initiated concurrently with addition of 2.5% DSSto the drinking water. Preliminary tracking studies with fluorescentlylabeled bovine serum albumin following intraperitoneal (i.p.) injectionof protein demonstrated proteins reached the colon at 6 hours after i.p.delivery. Based on these results, 6 hr prior to addition of 2.5% DSS tothe drinking water mice were treated with 50 nmoles/kg SG-14 i.p. or 0.2mg/kg Gly2-GLP2 i.p. Six hours after the initial treatment the drinkingwater was changed to water containing 2.5% DSS. The mice were treatedwith 2.5% dextran sodium sulfate (DSS) in their drinking water for 6days. Treatments were continued with SG-14 or Gly2-GLP2 twice a day(b.i.d.) in the morning and evening (every 8 and 16 hr) with i.p.injections at 50 nmoles/kg. Fresh 2.5% DSS drinking water was preparedevery 2 days.

On day six, mice were fasted for four hours and then orally gavaged with600 mg/kg 4 KDa dextran labeled with fluorescein isothiocyanate (FITC)[4 KDa-FITC]. One hour after the 4 KDa-FITC gavage mice were euthanized,blood was collected and FITC signal was measured in serum. A significantincrease in 4 KDa-FITC dextran translocation across the epithelialbarrier was observed in vehicle treated DSS mice in comparison tountreated mice. Additionally, a significant reduction in 4 KDa-FITCdextran was observed in mice receiving DSS and treated with SG-14, ascompared to DSS mice treated with vehicle. The magnitude of 4 KDa-FITCdextran translocation observed for SG-14-treated mice was even lowerthan the positive control of Gly2-GLP2. Results are shown in FIG. 6 ,and are presented as mean SEM. The graph in FIG. 6 represents datapooled from the single experiment (n=10).

Effects of SG-14 on Inflammation Centric Readouts of Barrier Function ina Concurrent DSS Model of Inflammatory Bowel Disease

SG-14 was also assessed for its effects on the levels of LPS bindingprotein (LBP) in the blood of the DSS animal with and without SG-14administration. LPS binding protein (LBP), which has been linked toclinical disease activity in subjects with inflammatory bowel disease,was also measured by ELISA in the serum of mice tested in the DSS modeldescribed in Example 7. A significant increase in LBP concentration wasobserved in vehicle-treated DSS mice, as compared to untreated mice.Additionally, a significant reduction in LBP was observed in SG-14treated mice given DSS as compared to DSS mice treated with vehicle.Furthermore, SG-14 had a greater impact on LBP concentration as comparedto the control peptide Gly2-GLP2, as a significant difference betweenDSS mice treated with Gly2-GLP2 and DSS mice treated with SG-14 wasobserved. Results are shown in FIG. 7 , and are presented as mean f SEM.The graph in FIG. 7 represents data pooled from the single experiment(n=10).

Effects of SG-14 on Weight Loss in a Concurrent DSS Model ofInflammatory Bowel Disease

Also assessed was the therapeutic ability of a protein as disclosedherein to ameliorate weight loss in an animal suffering from aninflammatory intestinal disorder. Weight loss is a significant andpotentially dangerous side effect of inflammatory bowel disease.

Body weight was measured daily from mice included in the DSS modeldescribed in this Example. Percent change from starting weight on day 0was determined for each mouse. SG-14 administration to DSS treated micesignificantly improved body weight as compared to vehicle treated DSSmice. Weight loss in mice treated with SG-14 at day 6 was similar toweight loss observed with vehicle. Results are shown in FIG. 8 . Thegraph in FIG. 8 represents data pooled from the single experiment(n=10).

Effects of SG-14 on Gross Pathology in a DSS Model of Inflammatory BowelDisease

Gross pathology observations were made in mice included in theconcurrent DSS model performed in this Example. SG-14 administration toDSS treated mice significantly improved gross pathology as compared tovehicle treated DSS mice. No differences, however, were observed betweenclinical scores in DSS mice treated with Gly2-GLP2 and DSS mice treatedwith SG-14. The scoring system used was: (0)=no gross pathology,(1)=streaks of blood visible in feces, (2)=completely bloody fecalpellets, (3) bloody fecal material visible in cecum, (4) bloody fecalmaterial in cecum and loose stool, (5)=rectal bleeding. Results areshown in FIG. 9 . The graph in FIG. 9 represents data pooled from thesingle experiment (n=10).

Effects of SG-14 on the Colon Shortening Effect in Response to DSSTreatment

The following experiment demonstrates the effects of SG-14 on colonshortening to treat in an in vivo model of inflammatory bowel disease.

Colon length was measured in mice included in the DSS model described inExample 7. SG-14 administration to DSS treated mice prevented colonshortening elicited by DSS treatment. A significant improvement in colonlength was observed with Gly2-GLP2 as a positive control. Results areshown in FIG. 10 . Results are shown in FIG. 10 . Also, a significantreduction of the ratio between colon weigh and length was observed inSG-14 administration to DSS treated mice along with Gly2-GLP2administration as shown in FIG. 11 . The graphs in FIG. 10 and FIG. 11represent data pooled from the single experiment (n=10).

Effects of SG-14 on Colonic Tissue Pathology Induced by DSS Treatment

The following experiment demonstrates the therapeutic effects of SG-14as disclosed herein on tissue pathology as assessed by histopathology.

Colon tissue was collected from mice in the DSS model described in thisExample 7. Tissues were fixed in 10% formalin, processed for paraffinembedding and sectioned. Tissue sections were stained with Hematoxylinand Eosin and scored for pathology by a pathologist specializing ingastrointestinal disease. The scoring system used multiple pathologyreadouts including inflammation, edema, transmural inflammation, mucosalhyperplasia, dysplasia, loss of mucosal architecture, the extent oftissue affected, and the extent of tissue affected by the most severepathology. Each readout was scored on a scale of 0-4 and a total scorewas generated by totaling all readout values for each treatment. SG-14administration to DSS treated mice significantly reduced colonicpathology elicited by DSS. SG-14 treatment also further reduced colonpathology scores significantly as compared to the positive controlGly2-GLP2 treatment. Results are summarized in FIG. 12A. Representativehistopathology images at 100× final magnification are shown in FIG. 123. The graph in FIG. 12A and images in FIG. 123 represent data from thesingle experiment (n=10).

Table 2 describes SEQ ID NOs of the present disclosure with detailedinformation.

TABLE 2 SEQ ID NO Type Description Name 1 PRT Full-length protein withsignal sequence SG-14 2 DNA coding sequence (cds) for SEQ ID NO: 1 3 PRTAmino acid sequence corresponding to amino SG-14 acid residues of 25 to655 of SEQ ID NO: 1 4 DNA cds for SEQ ID NO: 3 5 PRT SEQ ID NO: 3 with“start methionine” SG-14 6 DNA cds for SEQ ID NO: 5 7 PRT SEQ ID NO: 1without signal sequence SG-14 8 DNA cds for SEQ ID NO: 7 9 PRT Flag tag

NUMBERED EMBODIMENTS OF THE DISCLOSURE

Notwithstanding the appended claims, the disclosure sets forth thefollowing numbered embodiments:

Methods of Treatment

-   -   1. A method of treating a gastrointestinal epithelial cell        barrier function disorder, comprising:        -   a. administering to a patient in need thereof a            pharmaceutical composition, comprising:            -   i. a therapeutic protein comprising an amino acid                sequence having at least about 85% sequence identity to                SEQ ID NO: 3; and            -   ii. a pharmaceutically acceptable carrier.    -   2. The method of embodiment 1, wherein the gastrointestinal        epithelial cell barrier function disorder is a disease        associated with decreased intestinal epithelium integrity.    -   3. The method of embodiment 1 or 2, wherein the gastrointestinal        epithelial cell barrier function disorder is at least one        selected from the group consisting of: inflammatory bowel        disease, Crohn's disease, ulcerative colitis, pouchitis,        irritable bowel syndrome, enteric infections, Clostridium        difficile infections, metabolic diseases, obesity, type 2        diabetes, non-alcoholic steatohepatitis, non-alcoholic fatty        liver disease, liver disorders, alcoholic steatohepatitis,        celiac disease, necrotizing enterocolitis, gastro intestinal        disorders, short bowel syndrome, GI mucositis, chemotherapy        induced mucositis, radiation induced mucositis, oral mucositis,        interstitial cystitis, neurological disorders, cognitive        disorders, Alzheimer's, Parkinson's, multiple sclerosis, autism,        chemotherapy associated steatohepatitis (CASH), and pediatric        versions of the aforementioned diseases.    -   4. The method of any one of embodiments 1-3, wherein the        gastrointestinal epithelial cell barrier function disorder is        inflammatory bowel disease.    -   5. The method of any one of embodiments 1-4, wherein the        gastrointestinal epithelial cell barrier function disorder is        Crohn's disease.    -   6. The method of any one of embodiments 1-4, wherein the        gastrointestinal epithelial cell barrier function disorder is        ulcerative colitis.    -   7. The method of any one of embodiments 1-6, wherein the        therapeutic protein comprises an amino acid sequence having at        least about 90%, sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   8. The method of any one of embodiments 1-7, wherein the        therapeutic protein comprises an amino acid sequence having at        least about 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   9. The method of any one of embodiments 1-8, wherein the        therapeutic protein comprises an amino acid sequence having at        least about 97% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   10. The method of any one of embodiments 1-9, wherein the        therapeutic protein comprises an amino acid sequence having at        least about 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   11. The method of any one of embodiments 1-10, wherein the        therapeutic protein comprises an amino acid sequence having at        least about 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   12. The method of any one of embodiments 1-11, wherein the        therapeutic protein comprises an amino acid sequence selected        from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 5 and SEQ        ID NO: 7.    -   13. The method of any one of embodiments 1-12, wherein the        therapeutic protein comprises the amino acid sequence of SEQ ID        NO: 3.    -   14. The method of any one of embodiments 1-12, wherein the        therapeutic protein comprises the amino acid sequence of SEQ ID        NO:5.    -   15. The method of any one of embodiments 1-14, wherein the        administering comprises rectal, parenteral, intravenous,        topical, oral, dermal, transdermal, or subcutaneous        administration.    -   16. The method of any one of embodiments 1-15, wherein the        administering is to the: mouth, gastrointestinal lumen, and/or        intestines of the patient.    -   17. The method of any one of embodiments 1-16, wherein the        patient experiences a reduction in at least one symptom        associated with the gastrointestinal epithelial cell barrier        function disorder.    -   18. The method of any one of embodiments 1-17, wherein the        patient experiences a reduction in at least one symptom        associated with the gastrointestinal epithelial cell barrier        function disorder selected from the group consisting of:        abdominal pain, blood in stool, pus in stool, fever, weight        loss, frequent diarrhea, fatigue, reduced appetite, tenesmus,        and rectal bleeding.    -   19. The method of any one of embodiments 1-18, wherein the        administering reduces gastrointestinal inflammation in the        patient.    -   20. The method of any one of embodiments 1-18, wherein the        administering reduces intestinal mucosal inflammation in the        patient.    -   21. The method of any one of embodiments 1-18, wherein the        administering increases the production of mucin in intestinal        tissue in the patient.    -   22. The method of any one of embodiments 1-18, wherein        administering increases intestinal epithelium wound healing in        the patient.    -   23. The method of any one of embodiments 1-18, wherein        administering increases intestinal epithelial cell proliferation        in the patient.    -   24. The method of any one of embodiments 1-23, further        comprising: administering at least one second therapeutic agent        to the patient.    -   25. The method of any one of embodiments 1-24, further        comprising: administering at least one second therapeutic agent        to the patient, said second therapeutic agent selected from the        group consisting of: an anti-diarrheal, a 5-aminosalicylic acid        compound, an anti-inflammatory agent, an antibiotic, an        antibody, an anti-cytokine agent, an anti-inflammatory cytokine        agent, a steroid, a corticosteroid, and an immunosuppressant.

Pharmaceutical Compositions

-   -   1. A pharmaceutical composition, comprising:        -   a. a therapeutic protein comprising an amino acid sequence            having at least about 85% sequence identity to SEQ ID NO: 3,            SEQ ID NO: 5 and/or SEQ ID NO: 7; and        -   b. a pharmaceutically acceptable carrier.    -   2. The pharmaceutical composition of embodiment 1, wherein the        therapeutic protein comprises an amino acid sequence having at        least about 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   3. The pharmaceutical composition of any one of embodiments 1-2,        wherein the therapeutic protein comprises an amino acid sequence        having at least about 95% sequence identity to SEQ ID NO: 3, SEQ        ID NO: 5 and/or SEQ ID NO: 7.    -   4. The pharmaceutical composition of any one of embodiments 1-3,        wherein the therapeutic protein comprises an amino acid sequence        having at least about 97% sequence identity to SEQ ID NO: 3, SEQ        ID NO: 5 and/or SEQ ID NO: 7.    -   5. The pharmaceutical composition of any one of embodiments 1-4,        wherein the therapeutic protein comprises an amino acid sequence        having at least about 98% sequence identity to SEQ ID NO: 3, SEQ        ID NO: 5 and/or SEQ ID NO: 7.    -   6. The pharmaceutical composition of any one of embodiments 1-5,        wherein the therapeutic protein comprises an amino acid sequence        having at least about 99% sequence identity to SEQ ID NO: 3, SEQ        ID NO: 5 and/or SEQ ID NO: 7.    -   7. The pharmaceutical composition of any one of embodiments 1-6,        wherein the therapeutic protein comprises an amino acid sequence        selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO:        5 and SEQ ID NO: 7.    -   8. The pharmaceutical composition of any one of embodiments 1-7,        wherein the therapeutic protein comprises the amino acid        sequence of SEQ ID NO: 3.    -   9. The pharmaceutical composition of any one of embodiments 1-7,        wherein the therapeutic protein comprises the amino acid        sequence of SEQ ID NO: 5.    -   10. The pharmaceutical composition of any one of embodiments        1-9, formulated for rectal, parenteral, intravenous, topical,        oral, dermal, transdermal, or subcutaneous administration.    -   11. The pharmaceutical composition of any one of embodiments        1-10, formulated such that the therapeutic protein has activity        in the gastrointestinal lumen and/or intestines of the patient.

Expression Vectors

-   -   1. An expression vector, comprising: a polynucleotide, which        encodes a protein comprising an amino acid sequence having at        least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   2. The expression vector of embodiment 1, wherein the encoded        protein comprises an amino acid sequence having at least about        90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ        ID NO: 7.    -   3. The expression vector of any one of embodiments 1-2, wherein        the encoded protein comprises an amino acid sequence having at        least about 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   4. The expression vector of any one of embodiments 1-3, wherein        the encoded protein comprises an amino acid sequence having at        least about 97% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   5. The expression vector of any one of embodiments 1-4, wherein        the encoded protein comprises an amino acid sequence having at        least about 98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   6. The expression vector of any one of embodiments 1-5, wherein        the encoded protein comprises an amino acid sequence having at        least about 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   7. The expression vector of any one of embodiments 1-6, wherein        the encoded protein comprises an amino acid sequence selected        from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 5 and SEQ        ID NO: 7.    -   8. The expression vector of any one of embodiments 1-7, wherein        the encoded protein comprises the amino acid sequence of SEQ ID        NO: 3.    -   9. The expression vector of any one of embodiments 1-7, wherein        the encoded protein comprises the amino acid sequence of SEQ ID        NO: 5.

Host Cells

-   -   1. A host cell, comprising: an exogenous polynucleotide, which        encodes a protein comprising an amino acid sequence having at        least about 85% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   2. The host cell of embodiment 1, wherein the encoded protein        comprises an amino acid sequence having at least about 90%, 95%,        97%, 98%, or 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5        and/or SEQ ID NO: 7.    -   3. The host cell of any one of embodiments 1-2, wherein the        encoded protein comprises an amino acid sequence having less        than 100% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or        SEQ ID NO: 7.    -   4. The host cell of embodiment 1 or 2, wherein the encoded        protein comprises an amino acid sequence selected from the group        consisting of: SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.    -   5. The host cell of any one of embodiments 1-4, wherein the        exogenous polynucleotide further encodes a host cell specific        signal sequence.    -   6. The host cell of any one of embodiments 1, 2, 4 and 5,        wherein the exogenous polynucleotide comprises a nucleic acid        sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to a nucleic acid sequence selected from the group        consisting of: SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.    -   7. The host cell of any one of embodiments 1-6, wherein the host        cell is a prokaryotic cell.    -   8. The host cell of any one of embodiments 1-7, wherein the host        cell is an Escherichia coli cell.    -   9. The host cell of any one of embodiments 1-6, wherein the host        cell is a eukaryotic cell.    -   10. The host cell of any one of embodiments 1-6 and 9, wherein        the host cell is a Chinese Hamster Ovary cell.    -   11. A method of producing a protein, comprising: culturing the        host cell of any one of embodiments 1-10, under conditions        sufficient for expression of the encoded protein.

Isolated Proteins

-   -   1. An isolated therapeutic protein, comprising: an amino acid        sequence having at least about 85% sequence identity to SEQ ID        NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7.    -   2. The isolated therapeutic protein of embodiment 1, comprising:        an amino acid sequence having at least about 90% sequence        identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ ID NO: 7.    -   3. The isolated therapeutic protein of any one of embodiments        1-2, comprising: an amino acid sequence having at least about        95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ        ID NO: 7.    -   4. The isolated therapeutic protein of any one of embodiments        1-3, comprising: an amino acid sequence having at least about        97% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ        ID NO: 7.    -   5. The isolated therapeutic protein of any one of embodiments        1-4, comprising: an amino acid sequence having at least about        98% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ        ID NO: 7.    -   6. The isolated therapeutic protein of any one of embodiments        1-5, comprising: an amino acid sequence having at least about        99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5 and/or SEQ        ID NO: 7.    -   7. The isolated therapeutic protein of any one of embodiments        1-6, comprising: an amino acid sequence selected from the group        consisting of: SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.    -   8. The isolated therapeutic protein of any one of embodiments        1-7, wherein the protein increases electrical resistance in an        in vitro transepithelial electrical resistance assay.    -   9. The isolated therapeutic protein of any one of embodiments        1-8, wherein the protein increases electrical resistance in an        in vitro transepithelial electrical resistance assay by at least        about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or        99%, as compared to the assay performed in the absence of the        protein.    -   10. The isolated therapeutic protein of any one of embodiments        1-9, wherein the protein increases electrical resistance in an        in vitro transepithelial electrical resistance assay, as        compared to a control of a kinase inhibitor.    -   11. The isolated therapeutic protein of any one of embodiments        1-9, wherein the protein increases electrical resistance in an        in vitro transepithelial electrical resistance assay, as        compared to a control of staurosporine or myosin light chain        kinase.

Synthetic Therapeutic Protein

-   -   1. A synthetic therapeutic protein, comprising: an amino acid        sequence having at least about 90%, 95%, 97%, 98%, or 99%        sequence identity to SEQ ID NO:3, SEQ ID NO: 5 or SEQ ID NO:7.    -   2. The protein of embodiment 1, wherein the amino acid sequence        is less than 100% identical to SEQ ID NO: 5.    -   3. The protein of embodiment 1 or 2, wherein the amino acid        sequence is less than 100% identical to SEQ ID NO: 7.    -   4. The protein of any one of embodiments 1-3, comprising: an        amino acid sequence having at least about 98% sequence identity        to SEQ ID NO: 5.    -   5. The protein of any one of embodiments 1-4, comprising: an        amino acid sequence having at least about 99% sequence identity        to SEQ ID NO: 5.    -   6. The protein of any one of embodiments 1-5, comprising: the        amino acid sequence of SEQ ID NO: 5.    -   7. The protein of any one of embodiments 1-6, wherein the        protein increases electrical resistance in an in vitro        transepithelial electrical resistance assay.    -   8. A pharmaceutical composition, comprising: the protein of any        one of embodiments 1-6 and a pharmaceutically acceptable        carrier.    -   9. A method of treating a gastrointestinal epithelial cell        barrier function disorder, comprising:        -   a. administering to a patient in need thereof a            pharmaceutical composition, comprising:            -   i. a therapeutic protein comprising an amino acid                sequence having at least about 85% sequence identity to                SEQ ID NO: 3 and/or SEQ ID NO: 5; and            -   ii. a pharmaceutically acceptable carrier.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

REFERENCES

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1. A method of treating a gastrointestinal epithelial cell barrier function disorder, comprising: a. administering to a patient in need thereof a pharmaceutical composition, comprising: i. an isolated therapeutic protein comprising an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3 and/or SEQ ID NO: 5; and ii. a pharmaceutically acceptable carrier.
 2. The method of claim 1, wherein the gastrointestinal epithelial cell barrier function disorder is a disease associated with decreased intestinal epithelium integrity.
 3. The method of claim 1, wherein the gastrointestinal epithelial cell barrier function disorder is at least one selected from the group consisting of: inflammatory bowel disease, Crohn's disease, ulcerative colitis, pouchitis, irritable bowel syndrome, enteric infections, Clostridium difficile infection, celiac disease, necrotizing enterocolitis, short bowel syndrome, GI mucositis, chemotherapy induced mucositis, radiation induced mucositis, oral mucositis, interstitial cystitis, and pediatric versions of the aforementioned diseases.
 4. The method of claim 1, wherein the gastrointestinal epithelial cell barrier function disorder is inflammatory bowel disease.
 5. The method of claim 1, wherein the gastrointestinal epithelial cell barrier function disorder is Crohn's disease.
 6. The method of claim 1, wherein the gastrointestinal epithelial cell barrier function disorder is ulcerative colitis.
 7. The method of claim 1, wherein the isolated therapeutic protein comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 3 and/or SEQ ID NO:
 5. 8. The method of claim 1, wherein the isolated therapeutic protein comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 3 and/or SEQ ID NO:
 5. 9. The method of claim 1, wherein the isolated therapeutic protein comprises the amino acid sequence of SEQ ID NO:
 3. 10. The method of claim 1, wherein the isolated therapeutic protein comprises the amino acid sequence of SEQ ID NO:
 5. 11. The method of claim 1, wherein administering comprises rectal, parenteral, intravenous, topical, oral, dermal, transdermal, or subcutaneous administration.
 12. The method of claim 1, wherein administering is to the: mouth, gastrointestinal lumen, and/or intestines of the patient.
 13. The method of claim 1, wherein the patient experiences a reduction in at least one symptom associated with the gastrointestinal epithelial cell barrier function disorder.
 14. The method of claim 1, wherein the patient experiences a reduction in at least one symptom associated with the gastrointestinal epithelial cell barrier function disorder selected from the group consisting of: abdominal pain, blood in stool, pus in stool, fever, weight loss, frequent diarrhea, fatigue, reduced appetite, tenesmus, and rectal bleeding.
 15. The method of claim 1, wherein administering reduces one or more of: gastrointestinal inflammation in the patient and intestinal mucosal inflammation in the patient.
 16. The method of claim 1, wherein administering increases one or more of the production of mucin in intestinal tissue in the patient, intestinal epithelium wound healing in the patient, and intestinal epithelial cell proliferation in the patient.
 17. The method of claim 1, further comprising: administering at least one second therapeutic agent to the patient, said second therapeutic agent selected from the group consisting of: an anti-diarrheal, a 5-aminosalicylic acid compound, an anti-inflammatory agent, an antibiotic, an antibody, an anti-cytokine agent, an anti-inflammatory cytokine agent, a steroid, a corticosteroid, and an immunosuppressant.
 18. A pharmaceutical composition, comprising: a. an isolated therapeutic protein comprising an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3 and/or SEQ ID NO: 5; and b. a pharmaceutically acceptable carrier.
 19. An expression vector, comprising: a polynucleotide, which encodes a protein comprising an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3 and/or SEQ ID NO:
 5. 20. The expression vector of claim 19, wherein the amino acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to SEQ ID NO:3 or SEQ ID NO:5. 